Methods for tissue decellularization

ABSTRACT

A method for producing sterile, decellurized bioprosthetic tissue comprising: (a) contacting a human tissue with a hypotonic solution to produce a lysed tissue, (b) contacting the lysed tissue with a first surfactant to produce a surfactant-treated tissue, (c) contacting the surfactant-treated tissue with a nuclease enzyme solution to produce an enzyme-treated tissue, (d) contacting the enzyme-treated tissue with a cleaning solution comprising a second surfactant, a chaotropic agent or a mixture thereof to produce a decellurized tissue and (e) contacting the decellurized tissue with a bioburden reducing agent to produce the final bioprosthetic tissue. Kits to be used in conjunction with said method, as well as, the bioprosthetic tissue produced via said method are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/580,367, filed Oct. 9, 2012, which claims the benefit of PCTApplication No. PCT/IB2011/001538, filed Feb. 25, 2011, which claims thebenefit of U.S. Provisional Application No. 61/308,872, filed Feb. 26,2010, the entire disclosure of which is hereby incorporated by referencein its entirety for all purposes.

FIELD

The disclosure relates generally to the field of bioengineering and inparticular to methods for producing decellularized, sterilized, andbioprosthetic tissues.

BACKGROUND

Tissue and organ transplantation is a rapidly growing therapeutic fieldas a result of improvements in surgical procedures, advancements inimmunosuppressive drugs and increased knowledge of graft/hostinteraction. There are numerous investigations underway directed towardthe engineering of improved transplantable tissue grafts, however, it isgenerally believed in the industry that ideal implants have yet to beproduced.

The use of decellularized tissue scaffolds have been explored as apossibility for tissue engineering, however, the existing approacheshave proven inadequate. References of interest include U.S. Pat. Nos.6,743,574; 5,336,616; Gratzer et al., Tissue Engineering,12(10):2975-2983 (2006); Woods et al., Biomaterials, 26:7339-7349(2005); Williams et al., Acta Biomaterialia, 5:993-1005 (2009); Liao etal., Biomaterials, 29:1065-1074 (2008); Wilson et al., Ann Thorac Surg,60:S353-S358 (1995); PCT App. Pub. No. WO2006/101885; U.S. Pat. No.7,318,998; Kitagawa et al., J Med Invest, 48(3-4):123-132 (2001);Kearney, Clin Dermatol, 23:357-364 (2005); Azar, Clin Sport Med,28:191-201 (2009); and Simon et al., Eur J Cardiothorac Surg,23:1002-1006 (2003)).

Despite major advancements in the field of biomedical engineering,modern tissue transplantation remains associated with complicationsincluding inflammation, degradation, scarring, contracture,calcification (hardening), occlusion, and/or rejection. Moreover,existing technologies for producing decellularized tissue scaffolds havefailed to sufficiently reduce the number of cells in the tissue to anadequate level. Therefore, there is a need in the art for methods ofproducing tissues that avoid or reduce the above-described drawbacks andthus have greater short- and long-term usability. There is also afurther need in the art for methods of producing sterile, decellularizedtissues.

SUMMARY

Disclosed herein is a method for producing a human bioprosthetic tissuecomprising: contacting a human tissue with a hypotonic solution toproduce a lysed tissue; contacting the lysed tissue with a firstsurfactant solution to produce a surfactant-treated tissue; contactingthe surfactant-treated tissue with a nuclease enzyme solution to producean enzyme-treated tissue; contacting the enzyme-treated tissue with acleaning solution comprising a second surfactant, a chaotropic agent, ora mixture thereof, to produce a decellularized tissue; and contactingthe decellularized tissue with a bioburden reducing agent solution toproduce the human bioprosthetic tissue. In some embodiments, the methodfurther comprises contacting one or more of the tissues with aphysiologically isotonic solution.

In some embodiments, the human tissue is human skin tissue, wherein thehypotonic solution comprises 10 mM Tris buffer, wherein the firstsurfactant solution comprises 1% (v/v) Triton X-100® (octyl phenoxypolyethoxyethanol), wherein the nuclease enzyme solution comprises RNaseand DNase, wherein the cleaning solution comprises 1% (v/v) tri-n-butylphosphate (TnBP), wherein the bioburden reducing agent solutioncomprises 1% (v/v) Peracetic Acid, wherein each step of the method isperformed separately from the other steps of the method, and whereineach step of the method is immediately followed by a rinsing step priorto the start of the next step of the method. In some embodiments, themethod further comprises sonicating one or more of the tissues.

In some embodiments, one or more of the solutions further comprises aprotease inhibitor. In some embodiments, the protease inhibitor is aserine protease inhibitor, a metalloprotease inhibitor, or a combinationthereof. In some embodiments, the protease inhibitor isethylenediaminetetraacetic acid (EDTA). In some embodiments, theprotease inhibitor is phenylmethanesulfonyl fluoride.

In some embodiments, one or more of the solutions further comprises abioburden reducing agent. In some embodiments, the bioburden reducingagent comprises penicillin, streptomycin, peracetic acid, ethanol, or acombination thereof.

In some embodiments, the hypotonic solution comprises one or moreorganic or inorganic buffers, one or more antibiotics or antimycotics,an alkaline pH, and wherein the osmolarity of the solution is maintainedas hypotonic to cells.

In some embodiments, the first surfactant solution comprises a saltselected from the group consisting of KCl and NaCl, one or more organicor inorganic buffers, one or more antibiotics or antimycotics, analkaline pH, one or more protease inhibitors, and 0.2-3% (v/v) of aanionic, non-ionic, zwitterionic or cationic detergent selected from thegroup consisting of Triton X-100, Triton X-200, Tween 20, Tween 80,sodium deoxycholate, CHAPS, sodium dodecyl sulfate (SDS),N-lauroyl-sarcosinate, Igepal CA630, and Sulfobetain-10 and -16). Insome embodiments, the first surfactant solution is a >1 M salinesolution. In some embodiments, the first surfactant solution comprisesan anionic surfactant. In some embodiments, the anionic surfactant isTriton X-100®.

In some embodiments, the nuclease enzyme solution comprises anendonuclease selected from the group consisting of DNAse, RNAse, andBenzonase, wherein the solution is prepared with a physiological bufferselected from the group consisting of Hanks' Balanced Salt Solution(HBSS), HEPES, Phosphate Buffered Saline (PBS), Tris-Buffered Saline(TBS), and wherein the solution is maintained at a pH of 6-8. In someembodiments, the nuclease enzyme is DNase, RNase, or a combinationthereof.

In some embodiments, the cleaning solution comprises 0.2-3% (v/v) of ananionic, non-ionic, zwitterionic or cationic detergent selected from thegroup consisting of Triton X-100, Triton X-200, Tween 20, Tween 80,sodium deoxycholate, CHAPS, sodium dodecyl sulfate (SDS),N-lauroyl-sarcosinate, Igepal CA630, and Sulfobetain-10 and -16 orTri-n-butyl phosphate (TnBP), one or more organic or inorganic buffers,one or more antibiotics or antimycotics, an alkaline pH, and wherein thesolution is prepared in either an aqueous or a 70% ethanol solvent. Insome embodiments, the cleaning solution further comprises TRIZMA® base.In some embodiments, the cleaning solution further comprises about 70%ethanol. In some embodiments, the cleaning solution further comprisestri-n-butyl phosphate (TnBP).

In some embodiments, the method further comprises carrying out one ormore steps at a temperature of between about 22° C. and 40° C. In someembodiments, the contacting of the enzyme-treated tissue with thecleaning solution is carried out at about 22° C.

In some embodiments, the human tissue is a human skin tissue. In someembodiments, the human tissue is a soft human tissue. In someembodiments, the soft human tissue is a heart valve, tendon, ligament,artery, vein, diaphragm, pericardium, fascia, dura mater, tympanicmembrane, aortic conduit, or cartilage. In some embodiments, the humantissue is human allogeneic skin.

In some embodiments, the decellularized tissue is substantiallydecellularized. In some embodiments, the decellularized tissue is atleast 90% decellularized. In some embodiments, the decellularized tissueis at least 95% decellularized. In some embodiments, the decellularizedtissue is characterized by a substantial absence of positive stainingfor cell nuclei. In some embodiments, the decellularized tissue ischaracterized by a substantial absence of cellular DNA. In someembodiments, the decellularized tissue is characterized by a substantialabsence of one or more immunogenic proteins. In some embodiments, theimmunogenic protein is HLA-DR or HLA-A,B,C.

In some embodiments, the bioprosthetic tissue is characterized by asubstantial absence of pathogens and spores. In some embodiments, thedecellularized tissue is characterized by greater than 70-80% reductionin cytoskeletal proteins levels. In some embodiments, the cytoskeletalproteins are vimentin, beta-actin, alpha-actin, myosin, tubulin, andvinculin. In some embodiments, the bioprosthetic tissue includes dermis.

Also described herein is a bioprosthetic tissue produced using one ormore methods described herein.

Also described herein is a tissue, wherein the tissue is substantiallyfree of nucleic acids. Also described herein is a tissue, wherein thetissue is substantially free of one or more major histocompatibilitymolecules (MHC). Also described herein is a tissue, wherein the tissueis substantially free of Staphylococcus bacteria. Also described hereinis a tissue, wherein the tissue is substantially free of Streptococcusbacteria. Also described herein is a tissue, wherein the tissue issubstantially free of Enterococcus bacteria. Also described herein is atissue, wherein the tissue is substantially free of Bacillus bacteria.Also described herein is a tissue, wherein the collagen structure of thetissue is not substantially altered following treatment using a methoddescribed herein as compared to a fresh control tissue.

In some embodiments, a tissue described herein is substantially free ofStaphylococcus aureus bacteria. In some embodiments, a tissue describedherein is substantially free of Streptococcus pyogenes bacteria. In someembodiments, a tissue described herein is substantially free ofEnterococcus bacteria. In some embodiments, a tissue described herein issubstantially free of Bacillus subtilis. In some embodiments, a tissueis substantially free of fungus.

In some embodiments, a tissue described herein is human skin.

In some embodiments, the nucleic acid is DNA or RNA. In someembodiments, the nucleic acid is RNA. In some embodiments, the nucleicacid is DNA. In some embodiments, a tissue described herein has lessthan 0.5 ng/mg dry weight of tissue of DNA. In some embodiments, atissue described herein has less than 0.5 ng/mg dry weight of tissue ofDNA as measured by a PicoGreen® DNA Assay.

In some embodiments, the MHC is HLA-DR. In some embodiments, a tissue issubstantially free of HLA-DR as measured by immunohistochemistry. Insome embodiments, the MHC is HLA-A,B,C. In some embodiments, a tissue issubstantially free of HLA-A,B,C as measured by immunohistochemistry.

In some embodiments, a tissue is substantially free of Vimentin asmeasured by immunohistochemistry. In some embodiments, a tissue issubstantially free of Beta-Actin as measured by immunohistochemistry.

In some embodiments, a tissue comprises elastin as measured by histologyusing Van Gieson stain. In some embodiments, a tissue comprises one ormore proteoglycans as measured by histology using Masson's Trichromestain.

In some embodiments, the collagen structure of a bioprosthetic tissue isnot substantially altered compared to a fresh control tissue as assessedby the thermal stability of collagen. In some embodiments, thedenaturation temperature of a bioprosethetic tissue is not substantiallyaltered compared to a fresh control tissue. In some embodiments, thedenaturation temperature of a bioprosthetic tissue is about 64 to 68° C.as measured by a Hydrothermal Isometric Tension (HIT) test.

Also described herein is a kit comprising one or solutions describedherein. In some embodiments, the kit comprises a hyptotonic solution. Insome embodiments, the kit comprises a first surfactant solution, anuclease enzyme solution. In some embodiments, the kit comprises acleaning solution. In some embodiments, the kit comprises a bioburdenreducing agent solution. In some embodiments, the kit includesinstructions for use of the kit and its contents. In some embodiments,the kit includes a sterile container. In some embodiments, the kitincludes labeling with directions for use. In some embodiments, the kitcomprises instructions for contacting a human tissue with one or more ofthe solutions described herein. In some embodiments, the kit furthercomprises a tissue described herein. In some embodiments, the tissue isan untreated tissue. In some embodiments, the kit further comprisesinstructions for performing a method described herein.

Also described herein is a kit, comprising a bioprosthetic tissuedescribed herein or a tissue described above. In some embodiments, thekit includes instructions for using the tissue. In some embodiments, thekit includes instructions for use of the kit and its contents. In someembodiments, the kit includes a sterile container. In some embodiments,the kit includes labeling with directions for use. In some embodiments,the kit further comprises instructions for transplanting the tissue intoa subject in need thereof. In some embodiments, the kit comprises ahyptotonic solution. In some embodiments, the kit comprises a firstsurfactant solution, a nuclease enzyme solution. In some embodiments,the kit comprises a cleaning solution. In some embodiments, the kitcomprises a bioburden reducing agent solution. In some embodiments, thekit comprises instructions for contacting a human tissue with one ormore of the solutions described herein. In some embodiments, the kitfurther comprises instructions for performing a method described herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. Flowchart outlining the steps in one embodiment of thedecellularization process for human skin.

FIG. 2. Histology of the fresh (left) and decellularized (right) humanskin allograft. Note preservation of collagen fibre matrix (pink;examples shown with white arrows) and complete lack of cell nuclei(blue; examples shown with black arrows) after decellularization.Hematoxylin and Eosin stain, 200×.

FIG. 3. Histology of the fresh (left) and decellularized (right) humanskin allograft. Note preservation of collagen (pink; examples shown withwhite arrows) and elastin (black; examples shown with black arrows)fibre matrix. Verhoeff-Van Gicson stain, 400×.

FIG. 4. Histology of the fresh (left) and decellularized (right) humanskin allograft. Tissue sections from fresh human skin (on the left)stained with Masson's Trichrome stain (stains cells nuclei (blue;examples shown with grey arrows), cell cytoplasm (red/purple; examplesshown with black arrows) and collagen, elastin and proteoglycans of theextracellular matrix (green; examples shown with white arrows) revealthe presence of numerous cells in both the epidermis (bottom ofmicrograph) and dermis (middle and top of micrograph) with intactcollagen, elastin, and proteoglycans. In contrast, afterdecellularization (on the right), only green staining remains indicatingthe presence of collagen, elastin, and proteoglycans in the absence ofcellular materials. Masson's Trichrome stain, 200×.

FIG. 5. Immunohistochemistry of fresh (left) and decellularized (right)human skin allograft for detection of cytoskeletal protein Vimentin.Tissue sections from fresh human skin (on the left) treated using amonoclonal antibody that specifically recognizes the cytoskeletalprotein Vimentin and peroxidase as a stain, shows the presence ofVimentin in cells indicated by the dark brown colorations (examplesshown with white arrows). In contrast, after decellularization (on theright), there was a substantial reduction in the amount and intensity ofdark brown staining indicating the removal of the cell cytoskeletalprotein Vimentin. Large areas devoid of any staining were seen withindecellularized skin and where staining was evident, the intensity andfrequency typically present is represented in the example shown on theright. Anti-Vimentin antibody, Peroxidase staining, 200×.

FIG. 6. Immunohistochemistry of fresh (left) and decellularized (right)human skin allograft for detection of cytoskeletal protein Beta-Actin.Tissue sections from fresh human skin (on the left) treated using amonoclonal antibody that specifically recognizes the cytoskeletalprotein Beta-Actin and peroxidase as a stain, shows the presence ofBeta-Actin in cells indicated by the dark brown colorations (examplesshown with white arrows). In contrast, after decellularization (on theright), there was a substantial reduction in the amount and intensity ofdark brown staining indicating the removal of the cell cytoskeletalprotein Beta-Actin. Large areas devoid of any staining were seen withindecellularized skin and where staining was evident, the intensity andfrequency typically present is represented in the example shown on theright. Anti-Beta-Actin antibody, Peroxidase staining, 100×.

FIG. 7. Composite images of Fresh (top) and Aqueous Decellularized(bottom) human skin stained for the cytoskeletal protein vimentin (1:80dilution) identified by dark grey/black coloration. The composite imageswere created from individual images taken at 100× magnification. Thecomposite reveals the very sparse and minimal staining afterdecellularization indicating substantial removal of the cytoskeletalprotein vimentin.

FIG. 8. Composite images of Fresh (bottom) and Ethanol Decellularized(top) human skin stained for the cytoskeletal protein vimentin (1:80dilution) identified by dark grey/black coloration. The composite imageswere created from individual images taken at 100× magnification. Thecomposite reveals the very sparse and minimal staining afterdecellularization indicating substantial removal of the cytoskeletalprotein vimentin.

FIG. 9. Composite images of Fresh (top) and Aqueous Decellularized(bottom) human skin stained for the cytoskeletal protein Beta-actin(1:4000 dilution) identified by dark grey/black coloration. Thecomposite images were created from individual images taken at 100×magnification. The composite reveals the very sparse and minimalstaining after decellularization indicating substantial removal of thecytoskeletal protein Beta-actin.

FIG. 10. Composite images of Fresh (bottom) and Ethanol Decellularized(top) human skin stained for the cytoskeletal protein Beta-actin (1:4000dilution) identified by dark grey/black coloration. The composite imageswere created from individual images taken at 100× magnification. Thecomposite reveals the very sparse and minimal staining afterdecellularization indicating substantial removal of the cytoskeletalprotein Beta-actin.

FIG. 11. Immunohistochemistry of fresh (left) and decellularized (right)human skin allograft for detection of Human Leukocyte Antigen HLA-A,B,C.Tissue sections from fresh human skin (on the left) treated using amonoclonal antibody that specifically recognizes the cell membraneprotein HLA-A,B,C and peroxidase as a stain, shows the presence ofHLA-A,B,C on cells indicated by the dark brown colorations (examplesshown with white arrows). In contrast, after decellularization (on theright), there is an absence of dark brown staining indicating theremoval of the HLA-A,B,C cell membrane protein. This is a directindicator of the treatment's ability to remove cell membrane materials.Anti-HLA-A,B,C antibody, Peroxidase staining, 400×.

FIG. 12. Immunohistochemistry of fresh (left) and decellularized (right)human skin allograft for detection of Human Leukocyte Antigen HLA-DR.Tissue sections from fresh human skin (on the left) treated using amonoclonal antibody that specifically recognizes the immunogenic cellmembrane protein HLA-DR and peroxidase as a stain, shows the presence ofHLA-DR on cells indicated by the dark brown colorations (examples shownwith white arrows). In contrast, after decellularization (on the right),there is an absence of dark brown staining indicating the removal of theimmunogenic HLA-DR cell membrane protein. This is a direct indicator ofthe treatment's ability to remove cell membrane materials and a majorprotein associated with allograft rejection Anti-HLA-DR antibody,Peroxidase staining, 200×.

FIG. 13. Composite images of Fresh (bottom) and Aqueous Decellularized(top) human skin stained using a monoclonal antibody (1:400 dilution)that specifically recognizes the cell membrane protein HLA-A,B,C andperoxidase as a stain. The presence of HLA-A,B,C on cells is indicatedby the dark grey/black coloration. In contrast, after decellularization(on the top), there is an absence of staining indicating the removal ofthe HLA-A,B,C cell membrane protein. The composite images were createdfrom individual images taken at 100× magnification.

DETAILED DESCRIPTION

Decellularization technology is a method that has the potential torevolutionize tissue allograft transplantation. Decellularizationinvolves extracting cellular materials (the source of immune responseand viruses) from allograft tissues leaving an intact, structurallynative extracellular matrix scaffold comprised of collagen, elastin, andproteoglycans. Moreover, the decellularization process as describedherein gives rise to safer and more sterile grafts by eliminatingbacteria and viruses.

The description relates to preferred aspects by way of illustrationonly. It should be noted that from the following discussion, alternativeaspects disclosed herein will be readily recognized as viablealternatives that can be employed without departing from the principlesof what is claimed. It should be noted that the language used herein hasbeen principally selected for readability and instructional purposes,and it can not have been selected to delineate or circumscribe theinventive subject matter. Accordingly, the present disclosure isintended to be illustrative, but not limiting, of the scope of claimedmethods.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “adecellularized tissue” includes a combination of two or moredecellularized tissues, and the like.

As used herein any reference to “one aspect” or “an aspect” means that aparticular element, feature, structure, or characteristic described inconnection with the aspect is included in at least one aspect. Theappearances of the phrase “in one aspect” in various places in thespecification are not necessarily all referring to the same aspect.

The terms “comprises,” “comprising,” “includes,” “including,” “has,”“having” or any other variation thereof, are intended to cover anon-exclusive inclusion. For example, a process, method, article, orapparatus that comprises a list of elements is not necessarily limitedto only those elements but can include other elements not expresslylisted or inherent to such process, method, article, or apparatus.Further, unless expressly stated to the contrary, “or” refers to aninclusive or and not to an exclusive or. For example, a condition A or Bis satisfied by any one of the following: A is true (or present) and Bis false (or not present), A is false (or not present) and B is true (orpresent), and both A and B are true (or present).

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base which is either a purine or pyrimidine.Deoxyribonucleic acid (DNA) in the majority of organisms is the geneticmaterial while ribonucleic acid (RNA) is involved in the transfer ofinformation contained within DNA into proteins. The terms “nucleicacid,” “nucleic acid molecule,” “nucleic acid fragment,” “nucleic acidsequence or segment,” or “polynucleotide” may also be usedinterchangeably with gene, cDNA, DNA and RNA encoded by a gene.

The terms “polypeptide,” “protein,” and “peptide” are usedinterchangeably herein. It is well-known in the art of proteinbiochemistry that amino acids, the ‘building blocks’ of proteins, haveparticular sizes and characteristics, such as charge, hydrophobicity andhydrophilicity. For example, nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and tyrosine. Polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid.

In certain aspects, the present disclosure provides for tissue that issubstantially free of some component (e.g., one or more cytoskeletalproteins, MHC molecules, or pathogens such as bacteria and viruses). Asused herein, the term “substantially free” means that the presence of aparticular component is either not detected using known assays, or if itis detected, it is only present in an amount that is in accordance withthe tissue regulations of the U.S. Food and Drug Administration (FDA) asset forth in Title 21 Code of Federal Regulations (CFR), Parts 1270 and1271, herein incorporated by reference. In various aspects,“substantially free” can include a tissue that is about 90%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% free of a component. Incertain aspects, “substantially free” means that the tissue iscompletely free of the component. In certain aspects, “substantiallyfree” means that the tissue is 100% free of the component.

As used herein, a tissue that is “substantially free of a pathogen” or“substantially free of pathogens” and similar terminology about aspecific pathogen or class of pathogens means a tissue that complieswith American Association of Tissue Banks (AATB) Standards for TissueBanking under K2.200.

The term “decellularization” refers to the removal and/or extraction ofcells and/or cellular components from a tissue.

In certain aspects, the present disclosure provides for tissue that issubstantially decellularized. As used herein “substantiallydecellularized” means that the tissue is 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% free of cells. In certainaspects, “substantially decellularized” means that the tissue iscompletely free of cells. In certain aspects, “substantiallydecellularized” means that the tissue is 100% free of cells.

The term “human bioprosthetic tissue” refers to a human tissue that hasbeen substantially decellularized. In some embodiments, the humanbioprosthetic tissue has also been sterilized.

The phrase “cellular component,” as used herein, refers to substancesthat constitute a portion of a cell, including cell membranes andmacromolecules that are normally found enclosed within a cell membrane,embedded within a cell membrane, or attached to a cell membrane.

Any terms not directly defined herein shall be understood to have themeanings commonly associated with them as understood within the art ofthe present disclosure. Certain terms are discussed herein to provideadditional guidance to the practitioner in describing the compositions,devices, methods and the like of aspects of the present disclosure, andhow to make or use them. It will be appreciated that the same thing canbe said in more than one way. Consequently, alternative language andsynonyms can be used for any one or more of the terms discussed herein.No significance is to be placed upon whether or not a term is elaboratedor discussed herein. Some synonyms or substitutable methods, materialsand the like are provided. Recital of one or a few synonyms orequivalents does not exclude use of other synonyms or equivalents,unless it is explicitly stated. Use of examples, including examples ofterms, is for illustrative purposes only and does not limit the scopeand meaning of the aspects of the present disclosure herein.

Methods

In certain aspects, the decellularization methods of the presentdisclosure involve a series of progressively more sterile wash stepsthat include variations between hypoosmotic and hyperosmotic conditions,surfactant, enzyme, and physiological buffer solutions. FIG. 1 providesa general flowchart that is representative of the methods of oneembodiment of the present disclosure. Each stage of the process isdesigned to sequentially break down and remove cellular components whilepreventing the degradation or disruption of extracellular matrixcomponents.

According to certain aspects of the present disclosure, hypoosmoticconditions are first applied to burst cells. When the sample comprisesskin, these conditions also serve to gently remove the epidermalcellular layer while leaving the underlying basement membrane matrixintact. In certain aspects, a second solution is applied, wherein thatsolution comprises a high salt content and surfactant (e.g.,Triton-X100®). This second solution is utilized to solubilize and removecytoplasmic and cytoskeletal components. In further aspects, cellulargenetic materials are then degraded using endonucleases (e.g., DNAse,RNAse) which can also attack bacteria and viruses. In further aspects,tissues can subsequently be treated with a solution comprising achaotropic agent and/or a surfactant to further removed any residualdisrupted cellular materials. In certain aspects, that solutioncomprises tri-n-butyl phosphate (TnBP), a chaotropic agent that disruptshydrogen and hydrophobic bonds. TnBP is also known to improve sterilityby deactivating and removing viral proteins from blood products(Horowitz et al., Blood, 79(3):852-831 (1992)). Certain aspects of thepresent disclosure involve the additional step of washing the tissue assufficient to provide residual surfactant levels far below cellulartoxicity levels (Gratzer et al., Tissue Engineering, 12(10):2975-2983(2006)).

Although certain methods are asserted to provide tissuedecellularization, the present disclosure describes one of the firstknown methods actually giving rise to substantially completedecellularization. Most decellularization processes can be classified asa physically-based process, a chemically-based process, or abiochemically-based process. There are many protocols that borrow fromseveral of these process classifications and interpretation of theeffects of decellularization on the extracellular matrix becomes moredifficult as the complexity of the protocols increases.

The present disclosure relates in some aspects to a method of renderinga tissue acellular. In various aspects of the present disclosure,decellularization can be accomplished using a number of chemicaltreatments, including incubation in certain salts, detergents orenzymes. The method comprises exposing the tissue to a hypotonicsolution under conditions such that cell lysis results, and subjectingthe resulting tissue to nuclease treatment so as to remove nucleic acidsand associated phosphorous-containing groups. Nuclease treatmenteffectively stops cell replication and protein synthesis.

In some aspects, a method for producing a human bioprosthetic tissue isdescribed herein. In some aspects the method includes contacting a humantissue with a hypotonic solution to produce a lysed tissue. In someaspects the method includes contacting the lysed tissue with a firstsurfactant solution to produce a surfactant-treated tissue. In someaspects the method includes contacting the surfactant-treated tissuewith a nuclease enzyme solution to produce an enzyme-treated tissue. Insome aspects the method includes contacting the enzyme-treated tissuewith a cleaning solution comprising a second surfactant, a chaotropicagent, or a mixture thereof, to produce a decellularized tissue. In someaspects the method includes contacting the decellularized tissue with abioburden reducing agent solution to produce the human bioprosthetictissue.

In some aspects the method includes contacting a human tissue with ahypotonic solution to produce a lysed tissue. As used herein, “hypotonicsolution” means an aqueous solution with a salt concentration lower thanthat found in normal cells of the body (e.g., <300 mOsm). A hyptonicsolution is generally designed to (i) cause the cells in the tissue toabsorb water (hypotonic conditions) and eventually burst and (ii) causethe intact epidermal layer of a tissue to separate from the underlyingdermis while leaving the basement membrane intact. Thus, a hyptonicsolution can be any aqueous solution with a salt concentration less thanthat found in normal cells (i.e. <300 mOsm) that causes cell swelling. Ahyptonic solution can include one or more organic or inorganic buffers,an alkaline pH, one or more antibiotics/antimycotics, one or moreprotease inhibitors, and osmolarity maintained that is hypotonic tocells. In some aspects, alkaline pH helps to limit protease activity,where desired.

In some aspects the method includes contacting a human tissue with afirst surfactant solution to produce a surfactant-treated tissue. Asused herein “first surfactant solution” means a solution including ananionic, non-ionic, zwitterionic, and/or cationic detergent. An anionic,non-ionic, zwitterionic, and/or cationic detergent can include TritonX-100, Triton X-200, Tween 20, Tween 80, sodium deoxycholate, CHAPS,sodium dodecyl sulfate (SDS), N-lauroyl-sarcosinate, Igepal CA630,and/or Sulfobetain-10 and -16. In some aspects the first surfactantsolution can be a high saline (e.g., >1M), buffered solution at analkaline pH (e.g., pH=8-10) containing 0.2-3% (v/v) of the anionic,non-ionic, zwitterionic or cationic detergent. In some aspects the firstsurfactant solution can include salt (e.g., KCl, NaCl), one or moreorganic or inorganic buffers, one or more antibiotics/antimycotics, oneor more anionic, non-ionic, zwitterionic or cationic detergents and oneor more protease inhibitors. The first surfactant solution is generallydesigned to remove cell membranes and cytoskeletal components. AlkalinepH helps to limit protease activity, where desired.

The use of the detergent Triton X-100® has been demonstrated to removecellular membranes, as detailed in U.S. Pat. No. 4,801,299. Otheracceptable decellularizing detergents include polyoxyethylene (20)sorbitan mono-oleate and polyoxyethylene (80) sorbitan mono-oleate(TWEEN 20® and 80), sodium deoxycholate,3-[(3-chloramidopropyl)-dimethylammino]-1-propane-sulfonate,octyl-glucoside and sodium dodecyl sulfate. In some aspects, a detergentcan include octylphenol ethylene oxide condensate.

In some aspects the method includes contacting a human tissue with anuclease enzyme solution to produce an enzyme-treated tissue. As usedherein the term “enzyme” refers to a protein-based biocatalyst. Anenzyme can generally be used to breakdown nucleic and ribonucleic acids.Enzymes can include nucleases such as endonucleases (e.g., DNAse, RNAse,Benzonase). In some aspects, Deoxyribonuclease I is from bovine pancreas(Bos Taurus) (NM_174534.2; NP_776959.1). In some aspects, Ribonuclease Ais from bovine pancreas (Bos Taurus) (NM_181810.1; NP_861526.1).

In certain aspects, enzymes can be used to accomplish decellularization,including but not limited to dispase II, trypsin, and thermolysin. Theseenzymes react with different components of collagen and intercellularconnections in achieving their effects. Dispase II attacks Type IVcollagen, which is a component of the lamina densa and anchoring fibrilsof the basement membrane. Thermolysin attacks the bulbous phemphigoidantigen in the hemidesmosome of the basal layer of keratinocytes.Trypsin attacks the desmosome complex between cells. Due to theprotcolytic nature of these enzymes, care must be taken that cellularremoval occurs without significant damage to the extracellular matrix,including the basement membrane complex. This is a function ofconcentration, time and temperature. If used for too long a time or attoo high a concentration, dispase II for example can completely removethe basement membrane complex from the dermis.

In some aspects the method includes contacting a human tissue with acleaning solution comprising a second surfactant, a chaotropic agent, ora mixture thereof, to produce a decellularized tissue. As used hereinthe term “cleaning solution” means a solution that includes an anionic,non-ionic, zwitterionic and/or cationic detergent or a chaotropic agent.An anionic, non-ionic, zwitterionic and/or cationic detergent caninclude Triton X-100, Triton X-200, Tween 20, Tween 80, sodiumdeoxycholate, CHAPS, sodium dodecyl sulfate (SDS),N-lauroyl-sarcosinate, Igepal CA630, and/or Sulfobetain-10 and -16. Asused herein “chaotropic agent” means a substance that increases thetransfer of apolar groups to water because of its ability to decreasethe ‘ordered’ structure of water and to increase its lipophilicity. Achaotropic agent generally causes the dissolution of biologicalmembranes, the solubilization of particulate proteins, changes in thesecondary, tertiary, and quaternary structure of proteins, anddenaturation of nucleic acids. A chaotropic agent can includeTri-n-butyl phosphate (TnBP), ions (e.g. SCN⁻, CNS⁻, ClO₄ ⁻, I⁻, Br⁻,CH₃ ⁻, COO⁻), urea derivatives, guanidine derivatives. In some aspects acleaning solution can include one or more organic or inorganic buffers,one or more antibiotics/antimycotics, an alkaline pH, and the solutionis prepared in sterile water or 70% ethanol as the solvent. In someaspects a cleaning solution can include 0.2-3% (v/v) of an anionic,non-ionic, zwitterionic or cationic detergent or a chaotropic agent, oneor more organic or inorganic buffers, one or moreantibiotics/antimycotics, an alkaline pH, and the solution is preparedin either an (i) aqueous or (ii) 70% ethanol solvent. The cleaningsolution is generally designed to remove any remaining cellularcomponents (e.g., cytoskeletal proteins, DNA, RNA fragments). As above,alkaline pH helps to limit protease activity, where desired.

In some aspects the method includes contacting a human tissue with anisotonic solution. As used herein “isotonic solution” means an aqueoussolution with a salt concentration approximately equal or equal to thatfound in normal cells of the body. In some aspects, isotonic solutionscan include Lactated Ringer's solution, Normal Saline solution (0.9%),and Phosphate Buffered Saline (PBS).

In some aspects the method includes contacting a human tissue with aprotease inhibitor. As used herein “protease inhibitor” means an agentthat is capable of deactivating enzymes that are capable of degradingproteins. Protease inhibitors can include phenylmethanesulfonylfluoride(PMSF), aprotinin, leupeptin, and Ethylenediamineteteraacetic acid(EDTA).

In various aspects, protease inhibitors are employed in combination withother reagents to prevent degradation of the extracellular matrix.Collagen-based connective tissues contain proteases and collagenases asendogenous enzymes in the extracellular protein matrix. Additionally,certain cell types including smooth muscle cells, fibroblasts andendothelial cells contain a number of these enzymes inside vesiclescalled lysosomes. When these cells are damaged by events such ashypoxia, the lysosomes are ruptured and their contents released. As aresult, the extracellular matrix can undergo severe damage from protein,proteoglycan and collagen breakdown. This damage can be severe, asevidenced in clinical cases of cardiac ischemia where a reduction inoxygen which is insufficient to cause cell death results in pronounceddamage to the collagen matrix. Additionally, a consequence ofextracellular breakdown is the release of chemoattractants, whichsolicit inflammatory cells, including polymorphonuclear leukocytes andmacrophages, to the graft, which are intended to remove dead or damagedtissue. These cells also, however, perpetuate the extracellular matrixdestruction through a nonspecific inflammatory response. Accordingly,the processing solution contains one or more protease inhibitorsselected from the group of N-ethylmaleimide (NEM),phenylmethylsulfonylfluoride (PMSF) ethylenediamine tetraacetic acid(EDTA), ethylene glycol-bis-(2-aminoethyl(ether)NNN′N′-tetraacetic acid,ammonium chloride, elevated pH, apoprotinin and leupeptin to preventsuch damage.

In some aspects the method includes contacting a human tissue with abioburden reducing agent solution. As used herein “bioburden reducingagent” means an agent that can inactivate, destroy, or eliminateinfectious materials (e.g., bacteria, spores, fungi, mold, or viruses).Bioburden reducing agents can include an antibacterial agent, an alcohol(e.g., methyl, ethyl, propoyl, isopropyl, butyl, t-butyl), an antiviralagent, an antimycotic agent, chlorine dioxide, a detergent,antimicrobials, antifungal agents, hydrogen peroxide, sodium hydroxide,and/or peracetic acid. See also U.S. Pat. No. 5,460,962, hereinincorporated by reference.

The solutions of the present disclosure may optionally comprise anappropriate buffer. The buffer can involve one of many different organicbuffers. In certain aspects, an organic buffer is selected from thegroup consisting of 2-(N-morpholino)ethanesulfonic acid (MES), Tris(hydroxymethyl)aminomethane (TRIS) and(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES).Alternatively, a low salt or physiological buffer including phosphatebicarbonate acetate citrate glutamate with or without glycine, can bemore appropriate in certain applications.

In some aspects, one or more of the solutions used in the presentlydisclosed methods further comprise at least one protease inhibitor. Incertain aspects, the protease inhibitor is a serine protease inhibitor,metalloprotease inhibitor, or a combination thereof. In some aspects,one or more of the solutions used in the presently disclosed methodscomprises at least one enzyme. In certain aspects, that enzyme is DNase,RNase, or a combination thereof. In some aspects, one or more of thesolutions used in the presently disclosed methods comprises asurfactant. In certain aspects, that surfactant is an anionicsurfactant. In further aspects, the anionic surfactant is Triton X-100®.In some aspects, one or more of the solutions used in the presentlydisclosed methods further comprises TRIZMA® base. In further aspects,the present disclosure provides methods for producing a bioprosthetictissue, the method further comprising sonicating the tissue. In someaspects, one or more of the solutions used in the presently disclosedmethods further comprises tri-n-butyl phosphate (TnBP). In some aspects,one or more of the solutions used in the presently disclosed methodsfurther comprises about 70% ethanol.

In certain aspects, a combination of physical treatments and chemical orbiochemical treatments can be used for tissue decellularization.

In certain aspects, cells are lysed physically using osmotic gradients,mechanical compression/massage, or freeze-thaw cycles. Hypertonic andhypotonic treatments put hydrostatic pressure on cell membranes in anaqueous environment causing them to burst and release their cellcontents. The contents of the cell are then more accessible to latertreatments with detergents or isotonic washout procedures. Mechanicalcompression or massage can be used to encourage membrane degradation andgradually expose more cell membranes to extraction solutions.Freeze-thaw cycles can be used to kill cells and then fracture theircell membranes so that subsequent washout procedures can access internalcell contents and fragmented membranes.

In further aspects, the method includes carrying out one or more stepsat a temperature of between about 20° C. and 40° C. In further aspects,one or more steps of the method is carried out at about 30° C. Infurther aspects, one or more steps of the method is carried out at roomtemperature. In further aspects, one or more steps of the method iscarried out at 37° C. In further aspects, one or more steps of themethod is carried out at 22° C. In further aspects, one or more steps ofthe method is carried out at less than 20, about 20, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, about40, or more than 40° C.

In some aspects, tissues are harvested and prepared for treatment. Theextent of tissue preparation is dependant upon the tissue to be treated,but usually involves removing extraneous tissue elements and cutting thetissue to size in order to facilitate decellularization.

In some aspects, a tissue is immersed in a hypotonic salt (<300 mOsm)solution with a pH=7-9 (e.g., about 7, 7, about 8, 8, 9, or about 9),with anti-proteolytic agents whose concentration are dependant uponinhibitor used (e.g., phenylmethanesulfonylfluoride (PMSF), aprotinin,leupeptin, Ethylenediamineteteraacetic acid (EDTA)), anti-microbialagents (e.g., penicillin, vancomycin, streptomycin, gentamycin,kanamycin, neomycin, sodium azide (NaN₃)) with or without anti-fungalagents (e.g., Amphotericin B, Nystain) prepared with a type II cellculture grade deionized water. The tissue can be treated with a minimumvolume ratio of solution to tissue of 20:1, 30:1, 40:1, or 50:1 (e.g.,20:1-50:1), on a rotating shaker table at 40-65 RPM, for 24-48 hours,T=4-40° C., with solution changes occurring at 12 hour intervals.

In certain aspects, the tissue is then transferred to a high saline (>1M, NaCl, KCl) buffered solution (pH=8-10) containing 0.2-3% (v/v) of aanionic, non-ionic, zwitterionic or cationic detergent (e.g., TritonX-100®, Triton X-200®, TWEEN 20®, TWEEN 80®, sodium deoxycholate, CHAPS,sodium dodecyl sulfate (SDS), N-lauroyl-sarcosinate, Igepal CA630,Sulfobetain-10 and -16) and protease inhibitors (e.g.,phenylmethanesulfonylfluoride (PMSF), aprotinin, leupeptin,Ethylenediamineteteraacetic acid (EDTA)) prepared with a type II cellculture grade deionized water. The tissue is treated with a volume ratioof solution to tissue of 20:1, 30:1, 40:1, or 50:1 (e.g., 20:1-50:1), ona rotating shaker table at 40-65 RPM, for 24-48 hours, T=4-40° C., withsolution changes occurring at 12 hour intervals.

In further aspects, the tissue can then be subjected to rinses withsterile physiological buffer (e.g., Hanks' Balanced Salt Solution(HBSS), HEPES, Phosphate Buffered Saline (PBS), Tris-Buffered Saline(TBS)) pH=6-8, T=4-40° C., 5 minutes to 1 hour, and then treated with asolution of endonucleases (e.g., DNAse, RNAse, Benzonase) prepared in aphysiological buffer (e.g., Hanks' Balanced Salt Solution (HBSS), HEPES,Phosphate Buffered Saline (PBS), Tris-Buffered Saline (TBS)), pH=6-8,for 1-5 hours, T=20-40° C. Afterward, the tissue is rinsed in sterilephysiological buffer alone as specified above for 5 minutes-1 hour,T=4-40° C.

In additional aspects, the tissue is treated with a sterile solution of0.2-3% (v/v) anionic, non-ionic, zwitterionic or cationic detergent(e.g., Triton X-100®, Triton X-200®, TWEEN 20®, TWEEN 80®, sodiumdeoxycholate, CHAPS, sodium dodecyl sulfate (SDS),N-lauroyl-sarcosinate, Igepal CA630, Sulfobetain-10 and -16) orchaotropic agent (e.g., Tri-n-butyl phosphate (TnBP)) prepared in either(i) physiological buffer solution (Hanks' Balanced Salt Solution (HBSS),HEPES, Phosphate Buffered Saline (PBS), Tris-Buffered Saline (TBS))adjusted to pH=7-9 with anti-microbial agents (e.g., penicillin,vancomycin, streptomycin, gentamycin, kanamycin, neomycin, sodium azide(NaN₃)) with or without anti-fungal agents (Amphotericin B, Nystain) orwith (ii) 50-70% ethanol, for 24-48 hours at T=4-40° C. Afterward, thetissue is rinsed with a sterile 50 mM Tris buffer solution adjusted topH 9 (containing no detergent or chaotropic agent) for 12-24 hours atT=4-40° C.

In further aspects, the tissue is treated with a 0.05-3% (v/v) PeraceticAcid (PAA) solution in ethanol or Phosphate Buffered Saline (PBS)neutralized to pH=7 to 7.5, for a duration of 30 minutes to 4 hours atT=20-40° C. After, the tissue can be rinsed with sterile physiologicalbuffer (e.g., Hanks' Balanced Salt Solution (HBSS), HEPES, PhosphateBuffered Saline (PBS), Tris-Buffered Saline (TBS)) with anti-microbialagents (e.g., penicillin, vancomycin, streptomycin, gentamycin,kanamycin, neomycin, sodium azide (NaN₃)) with or without anti-fungalagents (e.g., Amphotericin B, Nystain) for 12-24 hours at T=4-40° C.

In further aspects, tissues can be bottled under sterile conditions ineither (i) sterile physiological buffer (e.g., Hanks' Balanced SaltSolution (HBSS), HEPES, Phosphate Buffered Saline (PBS), Tris-BufferedSaline (TBS)) with anti-microbial agents (e.g., penicillin, vancomycin,streptomycin, gentamycin, kanamycin, neomycin, sodium azide (NaN₃)) withor without anti-fungal agents (e.g., Amphotericin B, Nystain) or (ii)50-70% ethanol and stored at T=4-25° C.

In some aspects, human skin is procured and prepared for treatment. Theextent of tissue preparation is dependent upon the quality of the skinobtained, but usually involves removing extraneous tissue elements andcutting the tissue to size(s) required by end users. For this example,three specimens of human skin are treated in one container. The size ofthe specimens used is 4 cm×5 cm and tissues are treated with a volumeratio of solution to tissue of 50:1.

In some aspects, specimens are transferred to a lidded polypropylene jarcontaining a preferred volume of 300 mL of a hypotonic (<300 mmol/Lpreferred value of 10 mM) Tris buffer and a metalloprotease inhibitor ata concentration of 1 μM-25 mM (preferred value of 5 mM and inhibitorbeing used is ethylenediaminetetraacetic acid (EDTA)). This solution isadjusted to pH 7-9 (preferred at pH=8) using HCl/NaOH prior to use. Tothis container can be added antibiotics/antimycotics at 50-100 U/mL or50-100 μg/mL dependent upon agent (preferred using 5 mL/L of apenicillin/streptomycin 100× solution with a stock concentration of10,000 U/mL/10,000 mg/mL) and protease inhibitor (preferred value of0.35 mL/L of the serine protease inhibitor (5% phenylmethanesulfonylfluoride in 100% ethanol)). The container is allowed to mix gently on ashaker table at 40-65 RPM, T=20-25° C., for 24 hours with fresh solutionchanged at 12 hours. This stage is designed to (i) cause the cells inthe tissue to absorb water (hypotonic conditions) and eventually burstand (ii) cause the intact epidermal layer to separate from theunderlying dermis while leaving the basement membrane intact.

In some aspects, the previous hypotonic solution is decanted andreplaced with a minimum volume ratio of solution to tissue of 50:1(preferred 300 mL) of a high saline solution (preferred value of 1.5 Mof potassium chloride with 50 mM Tris buffer) containing 1% (v/v) TritonX-100 (octyl phenoxy polyethoxyethanol), metalloprotease inhibitor at aconcentration of 1-25 mM (preferred value of 5 mM of EDTA) and proteaseinhibitor (preferred value of 0.35 mL/L of the serine protease inhibitor(5% phenylmethanesulfonyl fluoride in 100% ethanol)). To the containeris added antibiotics/antimycotics at 50-100 U/mL or 50-100 μg/mLdependent upon agent (preferred using 5 mL/L of apenicillin/streptomycin 100× solution with a stock concentration of10,000 U/mL/10,000 mg/mL). The container is allowed to mix gently on ashaker table (at 40-65 RPM), T=20-25° C., for 36 hours with freshsolution changed every 12 hours. This stage of the treatment is designedto remove cell membranes and cytoskeletal components.

In some aspects, the previous high saline solution is decanted andreplaced with a volume ratio of solution to tissue of 50:1 (preferredvalue of 300 mL) of sterile deionized water. Specimens are rinsed for 30minutes. Following the rinse with sterile deionized water, it isreplaced with a volume ratio of solution to tissue of 50:1 (preferredvalue of 300 mL) of Hanks'/HEPES physiological buffer (0.14 M SodiumChloride, 5.4 mM Potassium Chloride, 0.26 mM Sodium Phosphate dibasic,0.44 mM Potassium Phosphate monobasic, 4.2 mM Sodium Bicarbonate, 10 mMHEPES Sodium salt, 8.3 mM Calcium Chloride dihydrate, 0.2 mM MagnesiumSulfate heptahydrate, and 0.25 mM Magnesium Chloride hexahydrate. Thissolution is adjusted to pH 7.35 using 2 M HCl/NaOH prior to use).Specimens are rinsed in physiological buffer for 30 minutes.

In some aspects, following this rinse, the physiological buffer isdecanted and replaced with 200 mL of Hanks'/HEPES physiological buffer.To this is added endonucleases, preferred DNAse and RNAse, at preferredquantities of 1330 μL of deoxyribonuclease stock (type II from bovinepancreas, 13.3 U/μL NaCl/glycerol pH 7.3 solution and 1330 μL ofribonuclease stock (type III-A from bovine pancreas, 85 μg/mL). Thetissue is then placed in a shaking water bath, gently mixed (45-60 RPM)at 37° C. for 5 hours. After 5 hours the solution is decanted andreplaced with fresh Hanks'/HEPES physiological buffer, briefly rinsingthe tissue. This stage of the process is designed to degrade DNA and RNAto facilitate their subsequent removal.

In some aspects, the previous physiological buffer rinse is decanted andreplaced by 300 mL of a 50 mM Tris buffer solution adjusted to pH 9 andcontaining 1% (v/v) Tri-n-butyl phosphate (TnBP). To the container isthen added 1.5 mL of a penicillin/streptomycin antibiotic/antimycotic100× solution (stock concentration of 10,000 U/mL/10,000 mg/mL). Thisstep of the decellularization treatment can also be performed using asolution made up as 1% (v/v) TnBP in 70% ethanol withoutantibiotics/antimycotics. The tissue then gently mixed on a shaker table(45-60 RPM) at T=20-25° C. for 48 hours with fresh solution changesevery 12 hours. This stage is designed to further remove any remainingcellular components (cytoskeletal proteins, DNA, RNA) as TnBP is a“surfactant like” chaotropic solvent. Further, TnBP has been shown todeactivate viruses.

In some aspects, after treatment, the solution is decanted and refreshedwith 300 mL of 50 mM Tris buffer solution adjusted to pH 9 (containingno TnBP). The same amount of antibiotic/antimycotic solution(penicillin/streptomycin) previous used is added and the container isallowed to mix gently on a shaker table (at 40-65 RPM), T=20-25° C., for24 hours with fresh solution changed at 12 hours.

In some aspects, the previous pH 9 Tris buffer solution is decanted andreplaced with 300 mL of a 1% (v/v) Peracetic Acid (PAA) solution inethanol (PAA solution consisted of 2% peracetic acid, 100% ethanol, andsterile water (ratio v/v/v 2/1/1), providing a final sterilizationsolution of 1% PAA) for 4 hours, mixing gently on a shaker table (at40-65 RPM), at T=20-40° C. After PAA treatment, fresh sterilephosphate-buffered saline solution (PBS, containing 0.14 M sodiumchloride, 2.7 mM potassium chloride, 6.5 mM sodium phosphate dibasic,1.5 mM potassium phosphate monobasic and is adjusted to pH 7.4). Thesame amount of antibiotic/antimycotic (penicillin/streptomycin) solutionused previously is added and the container is allowed to mix gently on ashaker table (at 40-65 RPM), T=20-25° C., for 12 hours. After, thespecimen is rinsed twice for 30 minutes each with 300 mL fresh sterilephosphate buffered saline solution at room temperature containing noantibiotics/antimycotics.

In some aspects, each piece of tissue is bottled under sterileconditions with a minimum volume ratio of solution to tissue of 20:1 to50:1 in either (i) fresh sterile phosphate buffered saline solutioninfused with penicillin/streptomycin (6 mL/L of 100× solution with astock concentration of 10,000 U/mL/10,000 mg/mL) solution or (ii) 70%ethanol and stored at 4° C.

Detergents

Critical to the success of many decellularization strategies aredetergents or surfactants. Various detergents can be used according tothe present methods. Detergents are useful for decellularization whenthey are added in a sufficient concentration to form micelles. A micelleis a cluster of detergent monomers, often spherical, that is oriented sothat the non-polar domains of the detergent molecules are interactinginternally, and the polar domains are interacting with water moleculesexternally. The concentration at which detergents form micelles iscalled the critical micelle concentration (CMC). The CMC varies withdecellularization conditions, including ionic strength, pH, temperatureand the presence of protein and lipids (including other detergentmolecules). Micelles occupy space in the aqueous environment, and assuch, must disrupt hydrogen bonds between water molecules. Duringdecellularization, the extraction of cell components, including lipidsand proteins from tissue, is accomplished when these micelles enter thetissue, dissolve non-polar components, and are washed out with solutionchanges.

Detergents can be classified by one of three designations: ionic,nonionic, and zwitterionic. Ionic detergents are either anionic orcationic, although cationic detergents are not used in decellularizationprocesses due to their strong denaturing tendencies. A subgroup of ionicdetergents are the bile acid salts, found in the intestine where theysolubilize fats. Bile acids are milder than other anionic detergentssuch as SDS. Nonionic detergents, such as Triton X-100®, have neutralpolar head groups and are non-denaturing to proteins. Nonionicdetergents break lipid-lipid and lipid-protein interactions. Finally,zwitterionic detergents, such as CHAPS, have properties of both ionicand nonionic detergents. Zwitterionic detergents are generally milderthan ionic detergents and more denaturing to proteins than nonionicdetergents.

Solvents

Various solvents can be used according to the present methods for thedisruption of protein and lipid interactions by destabilizinghydrophobic interactions. In some aspects, tri-n-butyl phosphate (TnBP)is used. An added benefit to the use of TnBP in a decellularizationprotocol is its proven anti-viral action (Horowitz et al., Blood,79:826-831 (1992); Horowitz et al., Dev Biol Stand, 81:147-161 (1993)).In further aspects of the present methods, any other solvent showinggood decellularization performance with very little damage to theextracellular matrix can be used (Woods, M Sc Thesis DalhousieUniversity, Halifax NS, Canada (2002); Woods et al., Biomaterials,26:7339-7349 (2005)).

Protease Protection and Antibiotic/Anti-Viral Treatments

During decellularization, lysed cells release the contents of lysosomesinto the extracellular space. Many of the enzymes released, especiallyproteases, are highly effective at degrading the extracellular matrix.One goal of decellularization is to preserve the intact extracellularmatrix while removing cellular components, so the action of degradativeenzymes is counterproductive. In various aspects of the presentdisclosure, protease inhibitors are present during the initial celllysis phase of the procedure. In further aspects of the presentdisclosure, protease inhibitors are additionally present duringsubsequent phases of the decellularization process.

According to the present disclosure, protease inhibition can be achievedthrough a combination of methods. In certain aspects, lysosomalproteases are inhibited by using elevated pH (˜pH 8). In furtheraspects, chelating agents that bind metallic enzyme cofactors such asmagnesium, iron, or zinc (e.g., EDTA or 1,10 phenanthroline) are alsoused. In various aspects, chemical inhibition of proteases can beachieved by the addition of any one of a number of common proteaseinhibitors. Chemical inhibitors according to the present methods caninclude phenylmethanesulfonylfluoride (PMSF), aprotinin, or leupeptin.PMSF (irreversible binding) and aprotinin (reversible binding) inhibitserine proteases such as trypsin and chymotrypsin, whereas leupeptin(low stability in aqueous environments) inhibits both serine andcysteine proteases.

According to certain aspects of the present disclosure, methods areprovided for the inhibition of bacteria. Inhibition of bacteria isbeneficial in part because the decellularization process is notgenerally performed aseptically. In various aspects, common antibioticsused for decellularization treatments are used and can include grampositive bacteria cell wall synthesis inhibitors (e.g., penicillin orvancomycin), bacteria and mycoplasma protein synthesis inhibitors (e.g.,streptomycin or gentamicin) and agents that induce ribosomal miscodingin gram positive/negative bacteria and mycoplasma (kanamycin). Infurther aspects, sodium azide (NaN₃) is also added to decellularizationsolutions to inhibit microbial growth.

In some aspects, one or more of the solutions used in the presentlydisclosed methods further comprises at least one bioburden reducingagent. In further aspects the bioburden reducing agent is penicillin,streptomycin, peracetic acid, or a combination thereof.

The use of allografts and xenografts is complicated by the potential fordisease transmission. Many porcine tissues contain porcine endogenousretrovirus (PERV), even after decellularization (Leyh et al., J ThoracCardiovasc Surg, 126:1000-1004 (2003); Walles et al., Eur J CardiothoracSurg, 24:358-363 (2003)). Thus, in certain aspects, methods are providedfor eliminating viruses. In further aspects, peracetic acid and/orethanol treatments are used in conjunction with decellularization forthe elimination of viruses (Hodde et al., Biotechnol Bioeng, 79:211-216(2002)). In further aspects, the organic solvent TnBP is employed forthe added purpose of diminishing the presence of viruses (e.g., humanimmunodeficiency virus (HIV)) (Horowitz et al., Blood, 79:826-831(1992); Horowitz et al., Dev Biol Stand, 81:147-161 (1993)). In furtheraspects, anionic detergents have the effect of diminishing the presenceof viruses (e.g., HIV and other enveloped viruses) (Luscher-Mattli,Antivir Chem Chemother, 11:249-259 (2000); Cserhati et al., EnvironmentInternational, 28:337-348 (2002)). In certain aspects, the extracellularmatrix is loaded with protease inhibitors, antibiotics, and antiviralagents at the end of the decellularization procedure.

Table A provides examples of a number of reagents and techniques thatcan be used according to the present disclosure along with theirrespective modes of action.

TABLE A Mode of Action PHYSICAL METHODS Osmotic Gradient Bursts orcontracts cells, disrupts cell membranes Freeze/Thaw Disrupts cellmembranes Mechanical Delamination Separates tissue layers along naturalplanes of dissection Agitation/Compression Increases exposure of cellmembranes to extraction solutions CHEMICAL METHODS Anionic DetergentsBile Acid Salt Sodium Deoxycholate Solubilizes phospholipids Disruptsprotcin-lipid interactions Generally non-denaturing, mild Synthetic SDSDisrupts protein-protein interactions (Sodium Dodecyl Sulfate) Denaturesand solubilizes proteins Binds virus particles (incl. HIV) TritonX-200 ® Solubilizes cellular components (alkylaryl polyether sulfonate)N-lauroyl-sarcosinate Ruptures cells Solubilizes cell membrane proteinsNonionic Detergents Triton X-100 ® Non-denaturing protein solubilization(PEG tert-octylphenyl ether) Tween 20 ® or 80 ® Solubilizes peripheralmembrane (PEG-sorbitan proteins monolaurate/oleate) Igepal CA630Non-denaturing protein solubilization (formerly sold as Nonidet P40)(Octylphenyl-PEG) N-octyl-β-D glucopyranoside Solubilization of membraneproteins Zwitterionic Detergents Sulfobetain-10 and -16 Help decreasemicelle size with anionic detergents Retain zwitterionic nature overwide pH range CHAPS Disrupts protein-protein interactionsNon-dcnaturing, forms small micelles Alcohols Glycerol Dcstroys bacteriaSolubilizes cell components Isopropanol Disrupts cell membranes,dissolves lipids Destroys bacteria and viruses Ethanol Destroys bacteria1-Butanol Extracts lipids Organic Solvents Tri(n-butyl)phosphate (TnBP)Disrupts protein-protein interactions, mild [65] Removes virus particles(incl. HIV) Acids HCl, H₂SO₄ Solubilize cells Peracetic Acid Destroysviruses, solubilizes cell remnants Bases NaOH Disrupt DNA/RNA, cellmembranes Destroy viruses, inactivates prions [167] NH₄OH Weakenslipid-protein bonds Chelators EDTA Bind to metallic cofactors,inhibiting enzymes EGTA Bind ions cells need to attach to substratesBIOCHEMICAL METHODS Membrane/Attachment Enzymes Trypsin Disruptsdesmosomes, focal adhesions Cleaves peptide bonds on Arg and Lys DISPASEAttacks type IV collagen, helps lift cells DISPASE II PhospholipaseDigests phospholipids in cell membranes Antigen-targeted EnzymesThermolysin Attacks antigen in the hemidesmosome of the basal layer ofkeratinocytes. α-galoctosidase Digests α-1,3-galactose antigenExonucleases DNAse I Facilitates hydrolysis of terminal DNA strandsRNAse A Facilitates hydrolysis of terminal RNA strands EndonucleasesBenzonase Facilitates degradation of internal bonds in DNA (single anddouble stranded) and RNA

Tissues suitable for use in the present method include those appropriatefor implantation. Tissues can be human in origin. The tissue isgenerally decellularized prior to any fixation. In certain aspects, thetissue may be a soft tissue. The decellularization method is applicableto various tissues, including heart valves, tendons, ligaments,arteries, veins, diaphragms, pericardium, fascia, dura mater, tympanicmembranes, aortic conduits, dermis, cartilage, or any other suitabletissue.

According to one aspect, the present disclosure provides methods forproducing a bioprosthetic tissue wherein the tissue is a human tissue.

According to one aspect, the present disclosure provides methods forproducing a bioprosthetic tissue wherein the tissue is a soft tissue. Infurther aspects, the soft tissue is a heart valve, tendon, ligament,artery, vein, diaphragm, pericardium, fascia, dura mater, tympanicmembrane, aortic conduit, dermis, or cartilage. In certain aspects, thetissue is dermis. In certain aspects, the tissue is human allogeneicskin.

Bioprostheses produced in accordance with the present disclosure can beused as replacements for defective tissues in mammals, particularlyhumans. Methods of effecting the replacement of, for example, heartvalves, tendons, ligaments, vessels, etc., are well known in the art.

In certain aspects, the present methods are applied to thedecellularization of skin. The current clinical use of partial thicknessallograft skin is limited to a temporary bandage for the treatment ofsevere burns. Immune response in the patient results in the rejection ofthe skin and need for additional treatment and grafting procedures. Thedecellularization process would allow for prolonged cover and even hostintegration and remodeling. The elimination of “bioburden” significantlyincreases the supply of available skin for transplant.

Decellularization Assays

The extent of decellularization can be determined histochemically, forexample, by staining the tissue with hematoxylin and eosin usingstandard techniques.

Immunohistochemical staining can also be used, for example, to visualizecell specific markers such as Beta-actin and histocompatibilityantigens—an absence of such markers being a further indication ofdecellularization. In certain aspects, immunohistochemical antibodystaining can be used to identify specific immunogens associated withrejection (e.g., HLA-DR) and the removal of cellular DNA below thedetection levels of current analysis methods.

In various aspects, the methods of the present disclosure yield tissue,wherein the tissue is characterized by a substantial absence of positivestaining for cell nuclei.

In various aspects, the methods of the present disclosure yield tissue,wherein the tissue is characterized by a substantial absence of cellularDNA.

In various aspects, the methods of the present disclosure yield tissue,wherein the tissue is characterized by a substantial absence ofimmunogenic proteins. In further aspects, the immunogenic protein isHLA-DR. In further aspects, the immunogenic protein is HLA-A,B,C.

In various aspects, the methods of the present disclosure yield tissue,wherein the tissue is characterized by more than 70-80, 70, 80, 90, 95,or 99% reduction in cytoskeletal proteins levels. In further aspects,the cytoskeletal proteins are vimentin, beta-actin, alpha-actin, myosin,tubulin, and vinculin.

Tissue

Tissues suitable for use in the present method include those appropriatefor implantation into humans. Tissues can be human in origin. The tissueis generally decellularized prior to any fixation or other use. Incertain aspects, the tissue may be a soft tissue. Tissues can includeskin, heart valves, tendons, ligaments, arteries, veins, diaphragms,pericardium, fascia, dura mater, tympanic membranes, aortic conduits,dermis, cartilage, or any other suitable tissue.

In some aspects, the tissue is a human tissue. In some aspects, thetissue is a soft tissue. In further aspects, the soft tissue is a heartvalve, tendon, ligament, artery, vein, diaphragm, pericardium, fascia,dura mater, tympanic membrane, aortic conduit, dermis, or cartilage. Incertain aspects, the tissue is dermis. In certain aspects, the tissue ishuman allogeneic skin.

Bioprostheses produced in accordance with the present disclosure can beused as replacements for defective tissues in mammals, particularlyhumans. Methods of effecting the replacement of, for example, heartvalves, tendons, ligaments, vessels, etc., are well known in the art.

In some aspects, a tissue is substantially free of nucleic acids. Insome aspects, a tissue is less than 90, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% free of nucleic acids. In some aspects the nucleic acidis RNA. In some aspects the nucleic acid is DNA. In some aspects, thetissue has less than 0.5 ng/mg (dry weight of tissue) of DNA. In someaspects, the tissue has less than 0.5 ng/mg (dry weight of tissue) ofDNA as measured by a PicoGreen® DNA Assay.

In some aspects, a tissue is substantially free of one or more majorhistocompatibility molecules (MHC). In some aspects, a tissue is lessthan 90, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% free of one ormore major histocompatibility molecules (MHC). In some aspects, the MHCis HLA-DR. In some aspects, the tissue is substantially free of HLA-DRas measured by immunohistochemistry. In some aspects, the MHC isHLA-A,B,C. In some aspects, the tissue is substantially free ofHLA-A,B,C as measured by immunohistochemistry.

In some aspects, a tissue is substantially free of one or more microbes.In some aspects, a tissue is less than 90, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100% free of one or more microbes. In some aspects, theone or more microbes include bacteria, viruses, and fungi. In someaspects, the one or more microbes is a bacteria. In some aspects thebacteria is a Staphylococcus aureus bacteria, a Streptococcus pyogenesbacteria, an Enterococcus bacteria, or a Bacillus subtilis bacteria. Insome aspects the bacteria is a Staphylococcus bacteria, a Streptococcusbacteria, an Enterococcus bacteria, or a Bacillus bacteria. In someaspects, the presence or absence of Bacillus is an accepted surrogatefor the presence or absence of Clostridium due its undesirableproperties and higher risk. In some aspects, sterility of the tissue isachieved based upon meeting a 6 log 10 reduction in bacterial speciesaccording to known international standards. In some aspects, the tissueis substantially free of fungus.

In some aspects, a tissue is substantially free of one or more cellularcomponents. In some aspects, a tissue is less than 50, 50, 60, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% free of one or morecellular components. In some aspects, the one or more cellularcomponents include one or more cytoskeletal proteins. In some aspects,the one or more cytoskeletal proteins can include vimentin, beta-actin,alpha-actin, myosin, tubulin, and/or vinculin. In some aspects, the oneor more cytoskeletal proteins include beta-actin. In some aspects, thetissue is substantially free of Beta-Actin as measured byimmunohistochemistry. In some aspects, the one or more cytoskeletalproteins include vimentin. In some aspects, the tissue is substantiallyfree of Vimentin as measured by immunohistochemistry.

In some aspects, the tissue comprises elastin. In some aspects, thetissue comprises elastin as measured by histology using Van Giesonstain. In some aspects, the tissue comprises one or more proteoglycans.In some aspects, the tissue comprises one or more proteoglycans asmeasured by histology using Masson's Trichrome stain.

In some aspects, the collagen structure of the tissue is notsubstantially altered compared to a fresh control tissue. In someaspects, the collagen structure of the tissue is not substantiallyaltered compared to a fresh control tissue as assessed by the thermalstability of collagen. In some aspects, the denaturation temperature ofthe tissue is not substantially altered compared to a fresh controltissue. In some aspects, the denaturation temperature of the tissue isabout 60, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, about 70, or 70° C. Insome aspects, the denaturation temperature of the tissue is measured bya Hydrothermal Isometric Tension (HIT) test.

Kits

In some embodiments, the invention further provides kits comprising thetissues and/or compositions described herein. In some embodiments, theinvention further provides kits comprising the tissues described herein.In some embodiments, a kit can include one or more of the solutionsdescribed herein. In some embodiments, a kit can include a hyptotonicsolution, a first surfactant solution, a nuclease enzyme solution, acleaning solution, and a bioburden reducing agent solution. In someembodiments, a kit can include an untreated tissue. In some embodiments,a kit can include instructions for decellularizing an untreated tissue.In some embodiments, a kit can include instructions for use one or moresolutions described herein. In some embodiments, a kit can includeinstructions for treating an untreated tissue with one or more of thesolutions described herein. In some embodiments, a kit can includeinstructions for using the solutions described herein in a methoddescribed herein.

In an embodiment, the invention provides a kit comprising a compositionof the invention, e.g., a composition comprising decellularized tissuein a solution, in an appropriately labeled container. In certainembodiments, such kits comprise one or more individually labeledcontainers containing unit-dosage or multi-dosage aliquots of thecomposition, useful for administering a defined amount of thecomposition to an individual. The kit can additionally compriseinstructions for administering the compositions of the invention to anindividual, including, e.g., instructions on the manner of transplantingthe tissue.

In an embodiment, the invention provides a kit comprising one or moresolutions of the invention in an appropriately labeled container. Incertain embodiments, such kits comprise one or more individually labeledcontainers containing unit-dosage or multi-dosage aliquots of the one ormore solutions, useful for preparing a defined amount of the tissue foruse in an individual. The kit can additionally comprise instructions forcontacting a tissue with one or more of the solutions. In someembodiments, a kit can include instructions for carrying out one or moreof the methods described herein, e.g., the methods described in theclaims as filed.

The container in which kit components are handled and sold can belabeled per applicable Food and Drug Administration standards.

Although the foregoing methods, kits, and compositions have beendescribed in detail by way of example for purposes of clarity ofunderstanding, it will be apparent to the artisan that certain changesand modifications are comprehended by the disclosure and can bepracticed without undue experimentation within the scope of the appendedclaims, which are presented by way of illustration not limitation.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of these embodiments of the present invention will employ,unless otherwise indicated, conventional methods of protein chemistry,biochemistry, recombinant DNA techniques and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.See, e.g., T. E. Creighton, Proteins: Structures and MolecularProperties (W.H. Freeman and Company, 1993); A. L. Lehninger,Biochemistry (Worth Publishers, Inc., current addition); Sambrook, etal., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); MethodsIn Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);Remington Pharmaceutical Sciences, 18th Edition (Easton, Pa.: MackPublishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry3^(rd) Ed. (Plenum Press) Vols A and B(1992).

Example 1: Decellularization of Human Skin

Materials and Methods

Decellularization Protocol

Fresh frozen human skin from 6 donors (n=6) was procured, thawed at roomtemperature in its packaging, and prepared for treatment. The extent oftissue preparation was dependent upon the quality of the skin obtained,but generally involved removing extraneous tissue elements and cuttingthe tissue to size. The size of skin used was 4 cm×5 cm and tissues weretreated with a minimum volume ratio of solution to tissue of 50:1 unlessotherwise stated. For this example, three specimens of human skin weretreated in one container. All chemicals were obtained from Sigma-Aldrichunless otherwise noted and the product numbers are provided in brackets.

The basic decellularization protocol followed is outlined in FIG. 1.This first stage of the decellularization treatment was designed to (i)cause the cells in the tissue to absorb water (hypotonic conditions) andeventually burst and (ii) cause the intact epidermal layer to separatefrom the underlying dermis while leaving the basement membrane intact.Specimens of fresh human skin were transferred to a lidded polypropylenejar containing 300 mL of a hypotonic solution containing 10 mM Trisbuffer (Trizma Base, T87602) and 5 mM ethylenediaminetetraacetic acid(EDTA, E9884) as a metalloprotease inhibitor. This solution was adjustedto pH=8 using HCl/NaOH prior to use. To this container was added 5 mL/Lof a penicillin/streptomycin 100× solution (A5955, stock concentrationof 10,000 U/mL/10,000 mg/mL) and 0.35 mL/L of a stock solution (5% w/vin 100% ethanol) of the serine protease inhibitor phenylmethanesulfonylfluoride (PMSF, P7626). The container was then allowed to mix gently ona shaker table at 40-65 RPM, T=20-25° C., for 24 hours with freshsolution changed at 12 hours. During the first 12 hours of treatment,the epidermis of the skin samples was released from the underlyingdermis and was removed from the container when the solution wasrefreshed.

This next stage of the treatment was designed to remove cell membranesand cytoskeletal components. The hypotonic solution was decanted andreplaced with 300 mL of a high saline solution containing 5 M potassiumchloride (KCl, P9541), 50 mM Tris buffer (T87602) containing 1% (v/v)Triton X-100 (octyl phenoxy polyethoxyethanol, T9284), and ametalloprotease inhibitor (5 mM of EDTA, E9884). This solution wasadjusted to pH=8 using HCl/NaOH prior to use. To this container wasadded 5 mL/L of a penicillin/streptomycin 100× solution (A5955, stockconcentration of 10,000 U/mL/10,000 mg/mL) and 0.35 mL/L of a stocksolution (5% w/v in 100% ethanol) of the serine protease inhibitorphenylmethanesulfonyl fluoride (PMSF, P7626). The container was allowedto mix gently on a shaker table (at 40-65 RPM), T=20-25° C., for 36hours with fresh solution changed every 12 hours.

To ensure sterility, all work from this point onward was carried out ina Class II/III biological safety cabinet using aseptic technique. Theprevious high saline solution was decanted and replaced with 300 mL ofsterile, deionized water. Specimens were rinsed for 30 minutes.Following the rinse, the sterile deionized water was decanted andreplaced with 300 mL of sterile Hanks'/HEPES physiological buffer (0.14M Sodium Chloride (S9625), 5.4 mM Potassium Chloride (P9541), 0.26 mMSodium Phosphate dibasic (S0876), 0.44 mM Potassium Phosphate monobasic(P5379), 4.2 mM Sodium Bicarbonate (S8875), 10 mM HEPES Sodium salt(H7006), 8.3 mM Calcium Chloride dihydrate (C3881), 0.2 mM MagnesiumSulfate heptahydrate (M1880), and 0.25 mM Magnesium Chloride hexahydrate(M0250). This solution was adjusted to pH 7.35 using HCl/NaOH prior touse. Specimens were rinsed in the Hanks'/HEPES physiological buffer for30 minutes.

The next stage of the process is designed to degrade DNA and RNA tofacilitate their subsequent removal. The Hanks'/HEPES physiologicalbuffer used for the rinse was decanted and replaced with 200 mL ofHanks'/HEPES physiological buffer containing 1330 μL ofdeoxyribonuclease stock solution (DNAse type II from bovine pancreas(D4527)), 13.3 U/μL, in a 0.175 g NaCl (S9625)/10 mL glycerol (G7757)/10mL sterile water solution adjusted to pH 7.3) and 1330 μL ofribonuclease stock solution (RNAse type III-A from bovine pancreas(R5125), 85 μg/mL, in a 0.03 g Trizma Base (T87602)/0.22 g NaCl(S9625)/25 mL sterile water solution adjusted to pH 7.5). The tissue wasthen placed in a shaking water bath, gently mixed (45-60 RPM) at 37° C.for 5 hours. After 5 hours the solution was decanted and replaced withfresh, sterile Hanks'/HEPES physiological buffer, briefly rinsing thetissue.

The next stage of the process was designed to further remove anyremaining cellular components (cytoskeletal proteins, DNA, RNA) as TnBPis a “surfactant like” chaotropic solvent. The previous physiologicalbuffer rinse was decanted and replaced by 300 mL of either (i) a 50 mMTris buffer (Trizma Base, T87602) solution in water adjusted to pH 9 andcontaining 1% (v/v) Tri-n-butyl phosphate (TnBP, 158615), 5 mL/L of apenicillin/streptomycin antibiotic/antimycotic 100× solution (stockconcentration of 10,000 U/mL/10,000 mg/mL) with samples denoted as“Aqueous Decellularization” or (ii) a 50 mM Tris buffer (Trizma Base,T87602) solution in 70% Ethanol adjusted to pH 9 and containing 1% (v/v)Tri-n-butyl phosphate (TnBP, 158615) with samples denoted as “EthanolDecellularization.” The samples of tissue were then gently mixed on ashaker table (45-60 RPM) at T=20-25° C. for 48 hours with fresh solutionchanges every 12 hours.

After treatment, the solutions were decanted and refreshed with 300 mLof 50 mM Tris buffer (Trizma Base, T87602) solution, adjusted to pH 9and containing 5 mL/L of a penicillin/streptomycinantibiotic/antimycotic 100× solution (stock concentration of 10,000U/mL/10,000 mg/mL). The container was then allowed to mix gently on ashaker table (at 40-65 RPM), T=20-25° C., for 24 hours with freshsolution changed at 12 hours.

Next, the previous pH 9 Tris buffer solution was decanted and replacedwith fresh, sterile, phosphate-buffered saline solution (PBS) containing0.14 M sodium chloride (NaCl, S9625), 2.7 mM potassium chloride (KCl,P9541), 6.5 mM sodium phosphate dibasic (S0876), 1.5 mM potassiumphosphate monobasic (P5379), adjusted to pH 7.4, and containing 5 mL/Lof a penicillin/streptomycin antibiotic/antimycotic 100× solution (stockconcentration of 10,000 U/mL/10,000 mg/mL). The container was allowed tomix gently on a shaker table (at 40-65 RPM), T=20-25° C., for 12 hours.After, the tissue specimens were rinsed twice (30 minutes each) with 300mL of fresh sterile phosphate buffered saline solution containing noantibiotics/antimycotics.

In a final step, each piece of tissue was bottled under sterileconditions in 100 ml of fresh sterile phosphate buffered saline (PBS)solution infused with penicillin/streptomycin (6 mL/L of 100× solutionwith a stock concentration of 10,000 U/mL/10,000 mg/mL), labeled aseither “Aqueous Decellularization” or “Ethanol Decellularization” andstored at 4° C. until used for histological, immunohistochemical, DNAcontent, and Hydrothermal Isometric Tension (HIT) analyses as outlinedbelow.

Histology

Histology samples were prepared from the central portion of each pieceof human skin (Fresh, Aqueous Decellularization, EthanolDecellularization). Samples were placed in 10% acetate buffered formalin(Sigma-Aldrich, HT50-1-128) for 48 hours, after which they weretransferred to 100% ethanol in preparation for paraffin embedding. Afterembedding, samples were sectioned using a microtome, with sectionthickness set at 5 μm. Sections were mounted on silinated glass slidesand dried for a minimum of two hours. After drying, slides weresubjected to a series of wash and staining steps using protocols forHematoxylin and Eosin (stains cell nuclei blue and collagen pink),Masson's Trichrome (stains cell nuclei blue/black, collagen andproteoglycans blue/green), and Verhoff-Van Gieson (stains cell nucleiblack, collagen red/pink, elastin fibres black). After these steps,sections were sealed and covered using glass coverslips. For eachsample, two sections were evaluated along their entire length under 10×,20× and 40× objectives, a thorough description of each slide wasrecorded and representative images captured with a digital camera.Decellularized human skin prepared using Aqueous Decellularization andEthanol Decellularization were compared to untreated Fresh human skin(control).

Immunohistochemistry

Formalin-fixed, paraffin-mounted tissue sections (5 μm thickness) wereprepared and dewaxed using xylenes and ethanol. Slides were washed inphosphate buffered saline (PBS), and antigen retrieval was carried outusing a citrate buffer (pH=6.1) and pressure cooker (HEIR). Afterantigen retrieval, samples were then placed in 2% hydrogen peroxide inPBS for 10 minutes for blocking of endogenous peroxidases. After a briefPBS wash, samples were blocked with Protein Block (Dako, X0909) for 10minutes in a humid chamber. Primary antibody (25 μL) was placed on eachsample and incubated for 18 hours in a humid chamber. Primary antibodiesincluded anti-vimentin (Dako, M7020, dilutions of 1:40, 1:80), anti-BetaActin (Sigma, A5316, dilutions of 1:2000, 1:4000), anti-HLA-DR (Dako,M0746, dilutions of 1:50, 1:100), and anti-HLA-A,B,C (Abeam, ab70328,dilutions of 1:200, 1:400). After incubation, samples were washed inPBS, and treated using a Universal LSAB+Kit/HRP (Dako, K0690) forrabbit/goat/mouse antibodies according to manufacturer's instructions.Samples were washed with PBS, and color was developed usingDAB+Chromogen+substrate buffer for 7 minutes. After a brief water wash,samples were counterstained using Mayer's Hematoxylin for 2 minutes,Scott's water for 2 minutes. Samples were then dehydrated (xylenes andethanol) and mounted with cytoseal and glass coverslips. For eachsample, two sections were evaluated along their entire length under 10×,20× and 40× objectives; a thorough description of each slide wasrecorded. Representative images at 20× and 40× and a complete scan ofone section at 10× were captured with a digital camera. A composite ofthe complete scan of a section was created by piecing togetherindividual images captured along the sample using the 10× objective.Decellularized human skin prepared using Aqueous Decellularization andEthanol Decellularization was compared to untreated Fresh human skinNegative controls included sections treated by omitting primary orsecondary antibodies.

DNA Quantification

The quantity of DNA present in Fresh human skin, Aqueous Decellularized,and Ethanol Decellularized treated human skin was determined using afluorescence-based assay. Representative tissue samples from eachtreatment condition were freeze-dried and weighed prior to the assay. Todisassociate the DNA from the tissue, a small piece of freeze-driedtissue (9-11 mg dry weight (wt)) from each sample was digested bypapain. Papain digest solution was made by preparing buffer solution (50ml of digestion buffer was prepared by combining 0.130 g ammoniumacetate (Sigma-Aldrich, A1542), 0.019 g Na₂EDTA-2H₂O (Sigma-Aldrich,E5134) and 0.015 g. DL-dithiothreitol (DTT) (Sigma-Aldrich, 43815) indistilled water with pH adjusted to 6.2 and adding lyophilized papain(Sigma-Aldrich, P4762) to a concentration of 1 mg/mL. Digest solutionwas added to each sample at a ratio of 100 μL per mg dry weight tonormalize the quantity of tissue per volume. Following digestion, eachsample was briefly centrifuged at 10,000 g to remove insoluble orundigested material. A small aliquot (50 μL) from each digested samplewas analyzed using the Quant-iT Picorgreen Assay Kit (Invitrogen, P7581)according to the manufacturer's instructions. Standard curves werecreated in the range of 0-1,000 ng/ml for Fresh skin and 0-12.5 ng/mlfor Aqueous Decellularized and Ethanol Decellularized skin. Thefluorescence was measured on a multiplate reader at an excitationwavelength of 485 nm and an emission wavelength of 528 nm. All readingswere performed in duplicate and in black 96-well plates to prevent‘cross-talk’ between the wells. Results were normalized to the dryweight of tissue analyzed for each sample.

Hydrothermal Isometric Temperature (HIT) Test

Samples were tested in a custom-built, multisample HIT apparatus, asdescribed by Lee et al. [Lee et al. (1995)]. Samples were tested ingroups, up to six at a time. Stainless steel spring clamps were used togrip samples (n=4) of Fresh, Aqueous Decellularized, and EthanolDecellularized skin 2.5 cm×0.5 cm), leaving cm of tissue between thegrips. An initial load of 50 g (0.5 N) was applied to each of thesamples, suspended in a 4-liter beaker containing distilled water. Thesamples were then heated at a rate of ≈1-2° C./min from room temperatureto an isotherm maximum temperature of 90° C. Temperatures were measuredusing a centrally located thermistor probe positioned at midsampleheight. Load data were acquired using custom-built strain-gaugedcantilever load cells. The load cells and temperature probe wereinterfaced with a conditioning amplifier, and both data acquisition andsystem control were accomplished via a personal computer equipped with a12-bit A/D, D/A DAQ board (Model NB-MIO-16L; National Instruments) andcustom-written software programmed using LabVIEW 7.1 software (NationalInstruments). Temperature-time-load data were generated throughout HITtesting. As the water in which the samples were immersed was heated fromambient temperature to the 90° C. maximum, load, temperature, and timedata were captured in 1° C. intervals. A key measure of collagen'shydrothermal stability is the temperature at which a triple helixdenatures, its denaturation temperature (Td). This is the temperature atwhich enough thermal energy has been transferred to the sample toovercome the energy barrier to uncoiling and allow change to a new,lower-energy conformation. In HIT testing, samples are held underisometric constraint such, that when enough thermal energy is added, thecollagen molecules within the sample become able to uncoil. Their actualuncoiling is prevented as the sample is being held under isometricconstraint and the energetic impetus to uncoil generates a tensile forcethat is recorded as a distinctive increase in the load on the sample.This creates an elbow in the load-temperature plot and the temperatureat which this elbow begins is termed the Td of the sample. Differentregions within a collagenous sample, and within an individual collagenmolecule for that matter, have varying degrees of thermal stability. TheTd value recorded in this work therefore is a function of the thermalstability of all of the load-bearing collagen in the sample duringtesting.

Statistics for HIT

Comparisons made between groups were conducted using a one-way ANOVA andthe Tukey-Kramer HSD test. Comparisons were made at 95% significance(α=0.05). Note that the resolution of the HIT test is 1° C. and thatchanges in Td from control (fresh) tissue of greater than or equal to 3°C. is deemed practically significant.

Results

Histology

Sections of fresh human skin stained with hematoxyin and eosin revealeddense packing of cell nuclei (dark blue) in the epidermis below thekeratinized layer and individual and clusters of cell nuclei throughoutthe dermis (FIG. 2). After decellularization by both Aqueous and EthanolDecellularization techniques, there was a complete absence of anystaining for cell nuclei as observed under 100×, 200× and 400×magnifications (FIG. 2). Collagen structure, stained pink by eosin,however, appeared well-preserved in all decellularized skin.

Fresh human skin sections stained with Verhoff-Van Gieson stain revealednumerous black cell nuclei in the non-keratinized epidermis, strongblack staining elastic fibres within the deeper dermis, and pinkcollagen matrix in the dermis (FIG. 3). After decellularization by allmethods, the cells in the epidermis were no longer visible, however therelative intensity of both the collagen (pink) and elastin fibres(black) in the dermis were similar to that of fresh skin (FIG. 3).

Masson's Trichrome staining of fresh human skin revealed the presence ofnumerous blue stained cell nuclei and red stained cell cytoplasm in theepidermis and dermis. Features such as blood vessels and residual hairfollicles could be observed. The dermis included blue/green stainedcollagen, elastin, and glycosaminoglycans (FIG. 4). Afterdecellularization by all treatments, no evidence of dark blue or redstaining indicating the presence of cells was present. Green staining,however, indicating the presence of collagen, elastin andglycosaminoglycans was evident (FIG. 4).

Immunohistochemistry

Staining of Fresh human skin for the cytoskeletal protein vimentin usingmonoclonal antibodies and peroxidase revealed positive stained cells asindividuals and clusters throughout the tissue (FIGS. 5, 7 and 8). Afterdecellularization by Aqueous or Ethanol Decellularization protocols,both the amount and intensity of positive staining for vimentin wassignificantly reduced (FIGS. 5, 7 and 8). Large regions devoid of anypositive staining for vimentin were present. Composite images ofcomplete sections of human skin before and after decellularizationcreated by taking overlapping images at 100× magnification give a senseof the overall level of removal of vimentin (FIGS. 7 and 8).

Fresh human skin stained using antibodies against the cytoskeletalprotein Beta-actin revealed strong positive staining throughout thenon-keratinized epidermis and strong positive staining of individual andclusters of cells throughout the dermis (FIGS. 6, 9 and 10). Afterdecellularization by Aqueous or Ethanol Decellularization protocols,both the amount and intensity of positive staining for Beta-actin wassubstantially reduced (FIGS. 5, 7 and 8). Large regions devoid of anypositive staining for Beta-actin were present. Composite images ofcomplete sections of human skin before and after decellularizationcreated by taking overlapping images at 100× magnification give a senseof the overall level of removal of Beta-actin (FIGS. 7 and 8).

Human Leukocyte Antigens (HLA) HLA-A,B,C and HLA-DR are majordeterminants of allograft skin rejection. HLA-A,B,C are present on allnucleated cells in the body and were shown to be present in thenon-keratinized epidermis and dermis in Fresh human skin byanti-HLA-A,B,C antibody and peroxidase staining (FIGS. 11 and 13).Staining was very intense and throughout the tissue. In contrast, nodetectable positive staining for HLA-A,B,C was present at 100×, 200×,and 400× magnification in any of the decellularized skin samples (FIG.11). HLA-DR, which is present on “antigen presenting cells” in the humanbody, was positively identified by anti-HLA-DR antibody and peroxidasestaining mostly in the dermis of Fresh skin samples (FIG. 12). Incontrast, no detectable positive staining for HLA-DR was present at100×, 200×, and 400× magnification in any of the decellularized skinsamples (FIG. 12). Composite images of complete sections of human skinbefore and after decellularization created by taking overlapping imagesat 100× magnification give a sense of the complete absence of positivestaining for HLA-A,B,C (FIG. 13). This also represented the effect ofdecellularization on the removal of HLA-DR and therefore a separatecomposite for HLA-DR was not shown.

DNA Quantification

DNA analysis of Fresh human skin using the Quant-iT Picorgreen Assay Kit(Invitrogen, P7581) revealed a content of 650±131 ng/mg (dry wt) oftissue. In contrast, no detectable amount of DNA was present afterdecellularization by Aqueous or Ethanol protocols (Table 1). Accordingto the manufacturer's directions, a lower detection level of <0.5 ng/mg(dry wt) was possible under our testing conditions.

Hydrothermal Isometric Temperature (HIT) Test

Samples of Fresh skin displayed a thermal denaturation temperature (Td)of 67.0±0.4° C. Decellularized skin processed by Aqueous (Td=67.1±0.8°C.) or Ethanol (Td=65.8±0.9° C.) protocols displayed similar thermaldenaturation temperatures (Table 2). This indicated that the structureof collagen within the skin was unaltered by our decellularizationmethods.

Conclusions

Both Aqueous and Ethanol decellularization protocols were able toproduce highly decellularized human skin. Analyses revealed that cellnuclei, major immunogenic cell proteins (HLA-DR and HLA-A,B,C), and DNAcontent were non-detectable using the methods described. Further,significant reductions in the cytoskeletal proteins vimentin andBeta-actin were achieved in decellularized skin. Finally,thermostability analysis of collagen revealed that the decellularizationmethods employed with human skin preserved the native structure ofcollagen within the tissue.

Example 2: Sterilization of Human Skin

Tissue banks process and store allografts by washing fresh tissues withantibiotic/antimycotic solutions followed by cryopreservation. Theseprocedures have proven inadequate for sterilization. Cryopreservationhas been shown by others to damage connective tissue structure [Kitagawaet al. (2001)]. Other sterilization techniques, such as ethylene oxideor gamma irradiation, have been shown to be deleterious to soft tissuegrafts and are not generally used by others [Kearney, J. N. (2005);Azar, F. M. (2009)].

According to AATB Standards for Tissue Banking under K2.200 “Skin shallnot be used for transplant if the following species are determined to bepresent: Staphylococcus aureus, Streptococcus pyogenes (group A strep.),Enterococcus sp., gram negative bacilli, Clostridium, and fungi (yeasts,molds)” [AATB (2006); (2008)]. In a study by Pianigiani et. al. (2009),they noted that the microorganisms responsible for skin allograftdiscard were all gram positives, the group of organisms most frequentlyisolated from pre-processing samples and included Staphylococcus,Streptococcus, Enterococcus, and Bacillus species. Four microorganismswere selected to determine if they can be removed and/or destroyed byour decellularization methods based on the American AATB Standards:Staphylococcus aureus, Streptococcus pyogenes (group A strep.),Enterococcus sp., and Bacillus subtilis which also was used as asurrogate for Clostridium due to its undesirable properties, such asodor and anaerobic culture conditions. Bacillus has been used as asurrogate for Clostridium in other studies [Stewart, Dunne, Skies, &Hoover, (2000)].

On the basis of on International Standards, ISO 11737-1 and 11737-2, aStandard Operating Procedure (SOP) of Bioburden Determination wasdeveloped in collaboration with professionals in the field ofMicrobiology. Preliminary experiments were first performed to validatethe use of tools and techniques.

Materials and Methods

Removing Inhibition from Fresh Frozen Skin

Human skin collected by tissue banks is stored in a cocktail ofantibiotics including Cefazolin and Gentomycin. To ensure completeremoval of any inhibitory effects of the storage antibiotic cocktail,samples of fresh frozen human skin with a broad range of storage timeswere tested (4-31 days of storage). Inoculants for the fourmicroorganisms used (S. aureus, Streptococcus, Enterococcus, andBacillus) were prepared by first obtaining a correlation between opticaldensity (OD) measured at 620 nm and the number of colony forming units(CFU)/mL. This was achieved by adjusting the OD value measured at 620 nmto 1 (acceptable range was 0.85 to 1.15) and spot plating the solutionto check the concentration. A concentration/dilution factor wascalculated from the correlation and used to make 10⁸ CFU/mL inoculantsfrom a one OD suspension of bacteria.

Sterile PBS rinse and vortexing were chosen as the methods to removeresidual antibiotics from skin allografts because these techniques hadminimal effect on the number of microorganisms attached to the skin. Thefrequency of such washing treatments was dependent upon the degree ofinhibition. Skin samples were subjected to different frequency of rinseswith 10 mL of PBS or three-minute vortexing with 20 mL of PBS. To checkthe presence of any inhibitory antibiotics after washing with PBS, 100μL of 10⁸ CFU/mL inoculants were added to a 50 mL centrifuge tubecontaining human skin and 10 mL sterile PBS. The tube was vortexed forone minute to allow release of antimicrobial substances from the skin.The working solution, which was already a 10⁻² dilution of the original10⁸ CFU/mL inoculants, was diluted by serial dilution. From the dilutedsolutions, six spots were made in Tryptic Soya Agar (TSA) plates bypipetting 100 μL of the solutions with 10⁻³ to 10⁻⁸ dilutions. Theinoculant solution was also directly spot plated with dilutions from10⁻³ to 10⁻⁸ for controls. Control plates of inoculants clearly showedthe number of viable bacteria that should grow in the absence ofinhibition.

Sterilization of Human Skin by Decellularization

Fresh frozen human skin from a total of four donors was procured, thawedat room temperature in its packaging, and prepared for treatment. Fromeach donor, fifteen 4 cm×5 cm pieces (corresponding to 3 samples foreach of the 4 microorganisms to be tested plus a negative control) andone 2 cm×5 cm (for bioburden background check) were prepared. Based uponthe results of our inhibition study, fresh human skin to be inoculatedwith S. aureus, Streptococcus or used as a negative control were vortexfor 3 minutes 6 times with 20 ml of sterile PBS; skin to be inoculatedwith Enterococcus, and Bacillus was vortex once with 20 mL of sterilePBS.

Inoculants (10⁸ CFU/mL) for the four microorganisms used, S. aureus,Streptococcus, Enterococcus, and Bacillus were prepared as describedpreviously under “Removing Inhibition from Fresh Frozen Skin” In a classII/III biological safety cabinet, a total of 4004 of each inoculantsolution was added to the dermal side of designated human skin samples(n=3 per microorganism) by pipetting 100 μL onto four quadrants andspreading evenly throughout the surface using a sterile transferpipette. As a negative control, 400 μl, of sterile PBS was added to 3samples of human skin. The inoculated tissues were then left to standunder laminar flow without drying out (60-75 minutes) to ensureabsorption of the bacterial inoculants. Samples of inoculated skin werethen transferred to separate 500 mL containers corresponding to S.aureus, Streptococcus, Enterococcus, Bacillus or the PBS negativecontrol with each container holding three 4 cm×5 cm samples. The samplesof skin were then treated with the Aqueous Decellularization or EthanolDecellularization, with or without Peractic Acid treatment as outlinedbelow.

Specimens of inoculated fresh human skin were first treated with 300 mLof a hypotonic solution containing 10 mM Tris buffer (Trizma Base,T87602) and 5 mM ethylenediaminetetraacetic acid (EDTA, E9884) as ametalloprotease inhibitor. This solution was adjusted to pH=8 usingHCl/NaOH prior to use. To this container was added 5 mL/L of apenicillin/streptomycin 100× solution (A5955, stock concentration of10,000 U/mL/10,000 mg/mL) and 0.35 mL/L of a stock solution (5% w/v in100% ethanol) of the serine protease inhibitor phenylmethanesulfonylfluoride (PMSF, P7626) The container was then allowed to mix gently on ashaker table at 40-65 RPM, T=20-25° C., for 24 hours with fresh solutionchanged at 12 hours. During the first 12 hours of treatment, theepidermis of the skin samples is released from the underlying dermis andis removed from the container when the solution is refreshed.

The hypotonic solution was decanted and replaced with 300 mL of a highsaline solution containing 5 M potassium chloride (KCl, P9541) 50 mMTris buffer (T87602) containing 1% (v/v) Triton X-100 (octyl phenoxypolyethoxyethanol, T9284) and a metalloprotease inhibitor (5 mM of EDTA,E9884) This solution was adjusted to pH=8 using HCl/NaOH prior to use.To this container was added 5 mL/L of a penicillin/streptomycin 100×solution (A5955, stock concentration of 10,000 U/mL/10,000 mg/mL) and0.35 mL/L of a stock solution (5% w/v in 100% ethanol) of the serineprotease inhibitor phenylmethanesulfonyl fluoride (PMSF, P7626). Thecontainer was allowed to mix gently on a shaker table (at 40-65 RPM),T=20-25° C., for 36 hours with fresh solution changed every 12 hours.

To ensure sterility, all work from this point onward was carried out ina Class II/III biological safety cabinet using aseptic technique. Theprevious high saline solution was decanted and replaced with 300 mL ofsterile deionized water. Specimens were rinsed for 30 minutes. Followingthe rinse, the sterile deionized water was decanted and replaced with300 mL of sterile Hanks'/HEPES physiological buffer (0.14 M SodiumChloride (S9625), 5.4 mM Potassium Chloride (P9541), 0.26 mM SodiumPhosphate dibasic (S0876), 0.44 mM Potassium Phosphate monobasic(P5379), 4.2 mM Sodium Bicarbonate (S8875), 10 mM HEPES Sodium salt(H7006), 8.3 mM Calcium Chloride dihydrate (C3881), 0.2 mM MagnesiumSulfate heptahydrate (M1880), and 0.25 mM Magnesium Chloride hexahydrate(M0250). This solution was adjusted to pH 7.35 using HCl/NaOH prior touse. Specimens were rinsed in the Hanks'/HEPES physiological buffer for30 minutes.

The Hanks'/HEPES physiological buffer used for the rinse was decantedand replaced with 200 mL of Hanks'/HEPES physiological buffer containing1330 μL of deoxyribonuclease stock solution (DNAse type II from bovinepancreas (D4527), 13.3 U/μL, in a 0.175 g NaCl (S9625)/10 mL glycerol(G7757)/10 mL sterile water solution adjusted to pH 7.3) and 1330 μL ofribonuclease stock solution (RNAse type III-A from bovine pancreas(R5125), 85 ng/mL, in a 0.03 g Trizma Base (T87602)/0.22 g NaCl(S9625)/25 mL sterile water solution adjusted to pH 7.5). The tissue wasthen placed in a shaking water bath, gently mixed (45-60 RPM) at 37° C.for 5 hours. After 5 hours the solution was decanted and replaced withfresh sterile Hanks'/HEPES physiological buffer, briefly rinsing thetissue.

The previous physiological buffer rinse was decanted and replaced by 300mL of a either (i) a 50 mM Tris buffer (Trizma Base, T87602) solution inwater adjusted to pH 9 and containing 1% (v/v) Tri-n-butyl phosphate(TnBP, 158615), 5 mL/L of a penicillin/streptomycinantibiotic/antimycotic 100× solution (stock concentration of 10,000U/mL/10,000 mg/mL) with samples denoted as “Aqueous Decellularization”or (ii) a 50 mM Tris buffer (Trizma Base, T87602) solution in 70%Ethanol adjusted to pH 9 and containing 1% (v/v) Tri-n-butyl phosphate(TnBP, 158615) with samples denoted as “Ethanol Decellularization”. Thesamples of tissue were then gently mixed on a shaker table (45-60 RPM)at T=20-25° C. for 48 hours with fresh solution changes every 12 hours.

After treatment, the solutions were decanted and refreshed with 300 mLof 50 mM Tris buffer (Trizma Base, T87602) solution, adjusted to pH 9and containing 5 mL/L of a penicillin/streptomycinantibiotic/antimycotic 100× solution (stock concentration of 10,000U/mL/10,000 mg/mL). The container was then allowed to mix gently on ashaker table (at 40-65 RPM), T=20-25° C., for 24 hours with freshsolution changed at 12 hours.

Next, the previous pH 9 Tris buffer solution was decanted and replacedwith 300 mL of either a (i) 1% (v/v) Peracetic Acid (PAA) solution inethanol (PAA solution consisted of 2% peracetic acid (269336), 100%ethanol, and sterile water (ratio v/v/v 2/1/1), providing a finalsterilization solution of 1% PAA) or (ii) directly into a fresh sterilephosphate-buffered saline (PBS) solution containing 0.14 M sodiumchloride (NaCl, S9625), 2.7 mM potassium chloride (KCl, P9541), 6.5 mMsodium phosphate dibasic (S0876), 1.5 mM potassium phosphate monobasic(P5379), adjusted to pH 7.4, and containing 5 mL/L of apenicillin/streptomycin antibiotic/antimycotic 100× solution (stockconcentration of 10,000 U/mL/10,000 mg/mL). The container was allowed tomix gently on a shaker table (at 40-65 RPM), T=20-25° C., for 12 hours.After, the tissue specimens were rinsed twice (30 minutes each) with 300mL of fresh sterile phosphate buffered saline solution containing noanitbiotics/antimycotics. For the Peracetic Acid treatment, samples weretreated for 4 hours, mixing gently on a shaker table (at 40-65 RPM), atT=20-40° C. After PAA treatment, the samples were placed in freshsterile phosphate-buffered saline (PBS) solution adjusted to pH 7.4, andcontaining 5 mL/L of a penicillin/streptomycin antibiotic/antimycotic100× solution (stock concentration of 10,000 U/mL/10,000 mg/mL). Thecontainer was allowed to mix gently on a shaker table (at 40-65 RPM),T=20-25° C., for 12 hours. After, the tissue specimens were rinsed twice(30 minutes each) with 300 mL of fresh sterile phosphate buffered salinesolution containing no anitbiotics/antimycotics.

Samples of Aqueous Decellularized and Ethanol Decellularized skin, withor without Peracetic Acid treatment were then immediately analyzed forthe presence of S. aureus, Streptococcus, Enterococcus, and Bacillus asoutlined below. Samples inoculated with sterile PBS only served as anegative control.

Detection and Enumeration of Bioburden

Samples (2×2.5 cm pieces) of human skin from each 500 mL containercorresponding to each decellularization method (Aqueous Decellularizedand Ethanol Decellularized skin, with or without Peracetic Acidtreatment) and microorganism inoculant (S. aureus, Streptococcus,Enterococcus, Bacillus or the PBS negative control) were prepared. Twosamples of skin per treatment, per microorganism inoculant, weredirectly plated on Tryptic Soya Agar (TSA) plates. One sample of skinper treatment, per microorganism inoculant was placed into a 50 mLpolypropylene tube for bacterial quantification. To the tube was added10 mL of sterile PBS and the tube sonicated for 5 minutes and Vortex for1 minute to release the bacteria. Dilutions of the supernatant werecreated (10⁻³ to 10⁻⁸) and spot plated onto TSA plates. The remaining“washed” skin was aseptically transferred onto TSA plates without excessliquid for direct plating. According to AATB guidelines [AATB (2006);(2008)], all plates were incubated at 37° C. for 7 days and the presenceor absence of bacterial colonies noted.

Further, to corroborate our findings, a 2×2.5 cm sample of human skinfrom each 500 mL container corresponding to each microorganism inoculant(S. aureus, Streptococcus, Enterococcus, Bacillus or the PBS negativecontrol) for decellularization Ethanol Decellularized skin withPeracetic Acid treatment were sent to the Department of Pathology andLaboratory Medicine, Capital District Health Authority, Halifax, NovaScotia, Canada for analysis according to the American Association ofTissue Banks (AATB) Standards (12^(th) edition) for human skin Testingincluded the detection of bacteria, yeast, or fungi. Samples wereincubated at 35° C. for 7 days for bacteria and at 30° C. for 30 daysfor yeast or fungi in their respective media, with readings taken on adaily basis. If positive growth was detected at any time, subculturingwas performed to identify the species of microbial contaminant.

As a positive control to show that decellularized skin was capable ofsupporting bacterial growth, samples of decellularized skin were alsoinoculated with each of the four species of bacteria used in this assay.The samples were then placed into a 50 mL polypropylene tube forbacterial quantification. To the tube was added 10 mL of sterile PBS andthe tube sonicated for 5 minutes and Vortex for 1 minute to release thebacteria. Dilutions of the supernatant were created (10⁻³ to 10⁻⁸) andspot plated onto TSA plates. The remaining “washed” skin was asepticallytransferred onto TSA plates without excess liquid for direct plating.The plates were incubated at 37° C. for 1 day and the presence orabsence of bacterial colonies noted.

Results

Controls

In all cases, negative controls (TSA plates alone, human skin inoculatedwith PBS) did not reveal the presence of bacterial colonies. Further,background level measurements for Staphylococcus aureus, Streptococcuspyogenes (group A strep.), Enterococcus sp., and Bacillus subtilisrevealed an absence of bacteria. For positive controls, decellularizedhuman skin inoculated with each of the four species of bacteria wasshown to be positive after only 1 day of incubation in both the washsolutions and by direct plating of the decellularized skin (Table 4).

Decellularization Methods

Decellularization of human skin by Aqueous and Ethanol protocols afterinoculation with 10⁸ CFU/mL of Staphylococcus aureus, Streptococcuspyogenes (group A strep.), Enterococcus sp., and Bacillus subtilis wasshown to eliminate all species but Bacillus subtilis (data not shown).When a 1% (v/v) peracetic acid treatment step was added to thedecellularization protocol, complete removal of all microbial specieswas obtained with a greater than 6 log 10 reduction in bacterial loadachieved as shown by the complete absence of bacteria in the washsolutions, by direct plating of the washed skin (labeled “Skin”), and bydirect plating of unwashed skin (labeled “Direct Plating”) (Table 3).

Table 5 shows the results of third-party testing (Department ofPathology and Laboratory Medicine, Capital District Health Authority,Halifax, Nova Scotia, Canada) for the presence of microorganismsaccording to the guidelines of the American Association of Tissue Banks(AATB). Decellularization of human skin by Ethanol Decellularizationprotocol with Peracetic acid treatment after inoculation with 10⁸ CFU/mLof Staphylococcus aureus, Streptococcus pyogenes (group A strep.),Enterococcus sp., and Bacillus subtilis was shown to completely removeall microbial species. In all samples, no bacteria or fungi weredetected.

Conclusions

Decellularization by Aqueous or Ethanol Decellularization protocols ontheir own did not achieve complete removal of all four species ofbacteria (Staphylococcus aureus, Streptococcus pyogenes (group Astrep.), Enterococcus sp., and Bacillus subtilis) used in inoculants onfresh skin. Complete removal of all four bacterial species was obtainedafter the inclusion of a 1% (v/v) peracetic acid rinse to thedecellularization protocols producing a human decellularized skin graftacceptable for transplant according to the American Association ofTissue Banks (AATB) standards [AATB (2006); (2008)].

Example 3: Decellularization and Sterilization of Human Fascia

Human fascia is procured and prepared for treatment. The extent oftissue preparation is dependent upon the quality of the fascia obtained,but usually involves removing extraneous tissue elements and cutting thefascia to size(s) required by end users. For this example, threespecimens of human fascia are treated in one container. The size of thespecimens used is 4 cm×5 cm and tissues are treated with a minimumvolume ratio of solution to tissue of 50:1. Specimens are transferred toa lidded polypropylene jar containing a preferred volume of 300 mL of ahypotonic (<300 mmol/L preferred value of 10 mM) Tris buffer and ametalloprotease inhibitor at a concentration of 1 μM-25 mM (preferredvalue of 5 mM and inhibitor being used is ethylenediaminetetraaceticacid (EDTA)). This solution is adjusted to pH 7-9 (preferred at pH=8)using HCl/NaOH prior to use. To this container is addedantibiotics/antimycotics at 50-100 U/mL or 50-100 μg/mL dependent uponagent (preferred using 5 mL/L of a penicillin/streptomycin 100× solutionwith a stock concentration of 10,000 U/mL/10,000 mg/mL) and proteaseinhibitor (preferred value of 0.35 mL/L of the serine protease inhibitor(5% phenylmethanesulfonyl fluoride in 100% ethanol)). The container isallowed to mix gently on a shaker table at 40-65 RPM, T=20-25° C., for24 hours with fresh solution changed at 12 hours. This stage is designedto cause the cells in the tissue to absorb water (hypotonic conditions)and eventually burst.

Next, the previous hypotonic solution is decanted and replaced with aminimum volume ratio of solution to tissue of 50:1 (preferred 300 mL) ofa high saline solution (preferred value of 1.5 M of potassium chloridewith 50 mM Tris buffer) containing 1% (v/v) Triton X-100 (octyl phenoxypolyethoxyethanol), metalloprotease inhibitor at a concentration of 1μM-25 mM (preferred value of 5 mM of EDTA) and protease inhibitor(preferred value of 0.35 mL/L of the serine protease inhibitor (5%phenylmethanesulfonyl fluoride in 100% ethanol)). To the container isadded antibiotics/antimycotics at 50-100 U/mL or 50-100 μg/mL dependentupon agent (preferred using 5 mL/L of a penicillin/streptomycin 100×solution with a stock concentration of 10,000 U/mL/10,000 mg/mL). Thecontainer is allowed to mix gently on a shaker table (at 40-65 RPM),T=20-25° C., for 36 hours with fresh solution changed every 12 hours.This stage of the treatment is designed to remove cell membranes andcytoskeletal components.

To ensure sterility, all work from this point onward is carried out in aClass II/III biological safety cabinet using aseptic technique. Theprevious high saline solution is decanted and replaced with a minimumvolume ratio of solution to tissue of 50:1 (preferred value of 300 mL)of sterile deionized water. Specimens are rinsed for 30 minutes.Following the rinse with sterile deionized water, it is replaced with aminimum volume ratio of solution to tissue of 50:1 (preferred value of300 mL) of Hanks'/HEPES physiological buffer (0.14 M Sodium Chloride,5.4 mM Potassium Chloride, 0.26 mM Sodium Phosphate dibasic, 0.44 mMPotassium Phosphate monobasic, 4.2 mM Sodium Bicarbonate, 10 mM HEPESSodium salt, 8.3 mM Calcium Chloride dihydrate, 0.2 mM Magnesium Sulfateheptahydrate, and 0.25 mM Magnesium Chloride hexahydrate. This solutionis adjusted to pH 7.35 using 2 M HCl/NaOH prior to use). Specimens arerinsed in physiological buffer for 30 minutes.

Following this rinse, the physiological buffer is decanted and replacedwith 200 mL of Hanks'/HEPES physiological buffer. To this is addedendonucleases, preferred DNAse and RNAse, at preferred quantities of1330 μL of deoxyribonuclease stock (type II from bovine pancreas, 13.3U/μL NaCl/glycerol pH 7.3 solution and 1330 μL of ribonuclease stock(type III-A from bovine pancreas, 85 μg/mL). The tissue is then placedin a shaking water bath, gently mixed (45-60 RPM) at 37° C. for 5 hours.After 5 hours the solution is decanted and replaced with freshHanks'/HEPES physiological buffer, briefly rinsing the tissue. Thisstage of the process is designed to degrade DNA and RNA to facilitatetheir subsequent removal.

The previous physiological buffer rinse is decanted and replaced by 300mL of a 50 mM Tris buffer solution adjusted to pH 9 and containing 1%(v/v) Tri-n-butyl phosphate (TnBP). To the container is then added 1.5mL of a penicillin/streptomycin antibiotic/antimycotic 100× solution(stock concentration of 10,000 U/mL/10,000 mg/mL). This step of thedecellularization treatment can also be performed using a solution madeup as 1% (v/v) TnBP in 70% ethanol without antibiotics/antimycotics. Thetissue then gently mixed on a shaker table (45-60 RPM) at T=20-25° C.for 48 hours with fresh solution changes every 12 hours. This stage isdesigned to further remove any remaining cellular components(cytoskeletal proteins, DNA, RNA) as TnBP is a “surfactant like”chaotropic solvent. Further, TnBP has been shown to deactivate viruses.

After treatment, the solution is decanted and refreshed with 300 mL of50 mM Tris buffer solution adjusted to pH 9 (containing no TnBP). Thesame amount of antibiotic/antimycotic solution (penicillin/streptomycin)previous used is added and the container is allowed to mix gently on ashaker table (at 40-65 RPM), T=20-25° C., for 24 hours with freshsolution changed at 12 hours.

Next, the previous pH 9 Tris buffer solution is decanted and replacedwith 300 mL of a 1% (v/v) Peracetic Acid (PAA) solution in ethanol (PAAsolution consisted of 2% peracetic acid, 100% ethanol, and sterile water(ratio v/v/v 2/1/1), providing a final sterilization solution of 1% PAA)for 4 hours, mixing gently on a shaker table (at 40-65 RPM), at T=20-40°C. After PAA treatment, fresh sterile phosphate-buffered saline solution(PBS, containing 0.14 M sodium chloride, 2.7 mM potassium chloride, 6.5mM sodium phosphate dibasic, 1.5 mM potassium phosphate monobasic and isadjusted to pH 7.4). The same amount of antibiotic/antimycotic(penicillin/streptomycin) solution used previously is added and thecontainer is allowed to mix gently on a shaker table (at 40-65 RPM),T=20-25° C., for 12 hours. After, the specimen is rinsed twice for 30minutes each with 300 mL fresh sterile phosphate buffered salinesolution at room temperature containing no anitbiotics/antimycotics.

In a final step, the each piece of tissue is bottled under sterileconditions with a minimum volume ratio of solution to tissue of 20:1 to50:1 in either (i) fresh sterile phosphate buffered saline solutioninfused with penicillin/streptomycin (6 mL/L of 100× solution with astock concentration of 10,000 U/mL/10,000 mg/mL) solution or (ii) 70%ethanol and stored at 4° C.

These processes produce decellularized and sterilized human fascia.Analyses similar to those employed in the Examples above and/or known inthe art reveal that cell nuclei, major immunogenic cell proteins (e.g.,HLA-DR and HLA-A,B,C), and DNA content are substantially reducedcompared to controls. Further, significant reductions in thecytoskeletal proteins such as, e.g., vimentin and Beta-actin, areachieved compared to controls. Thermostability analysis of collagenreveals that the decellularization methods largely preserve the nativestructure of collagen within the fascia. Decellularization by, e.g.,Aqueous or Ethanol Decellularization protocols, also reduces the amountof bacteria (Staphylococcus aureus, Streptococcus pyogenes (group Astrep.), Enterococcus sp., and Bacillus subtilis). Removal of all fourbacterial species is obtained via inclusion of a 1% (v/v) peracetic acidrinse. The resulting fascia graft is acceptable for transplant accordingto the guidelines provided by the American Association of Tissue Banks(AATB) standards [AATB (2006); (2008)].

Example 4: Decellularization and Sterilization of Soft Tissue

Tissue (e.g., a human soft tissue) is harvested and prepared fortreatment. The extent of tissue preparation is dependent upon the tissueto treated, but usually involves removing extraneous tissue elements andcutting the tissue to size in order to facilitate decellularization.Specimens are then immersed in a hypotonic salt (<300 mol/L) solution,pH=7-9, with anti-proteolytic agents whose concentration are dependentupon inhibitor used (phenylmethanesulfonylfluoride (PMSF), aprotinin,leupeptin, Ethylenediamineteteraacetic acid (EDTA)), anti-microbialagents (penicillin, vancomycin, streptomycin, gentamycin, kanamycin,neomycin, sodium azide (NaN₃)) with or without anti-fungal agents(Amphotericin B, Nystain) prepared with at minimum type II cell culturegrade deionized water. The tissue is treated with a minimum volume ratioof solution to tissue of 20:1 to 50:1, on a rotating shaker table at40-65 RPM, for 24-48 hours, T=4-40° C., with solution changes at minimumoccurring at 12 hour intervals.

The tissue is then transferred to a high saline (>1 M, NaCl, KCl)buffered solution (pH=8-10) containing 0.2-3% (v/v) of a anionic,non-ionic, zwitterionic or cationic detergent (Triton X-100, TritonX-200, Tween 20, Tween 80, sodium deoxycholate, CHAPS, sodium dodecylsulfate (SDS), N-lauroyl-sarcosinate, Igepal CA630, Sulfobetain-10 and-16) and protease inhibitors (candidates as listed above) prepared withat minimum type II cell culture grade deionized water. The tissue istreated with a minimum volume ratio of solution to tissue of 20:1 to50:1, on a rotating shaker table at 40-65 RPM, for 24-48 hours, T=4-40°C., with solution changes at minimum occurring at 12 hour intervals.

The tissue is then subjected to rinses with sterile physiological buffer(Hanks' Balanced Salt Solution (HBSS), HEPES, Phosphate Buffered Saline(PBS), Tris-Buffered Saline (TBS)) pH=6-8, T=4-40° C., 5 minutes to 1hour, and then treated with a solution of endonucleases (DNAse, RNAse,Benzonase) prepared in a physiological buffer (Hanks' Balanced SaltSolution (HBSS), HEPES, Phosphate Buffered Saline (PBS), Tris-BufferedSaline (TBS)), pH=6-8, for 1-5 hours, T=20-40° C. Afterward, the tissueis rinsed in sterile physiological buffer alone as specified above for 5minutes-1 hour, T=4-40° C.

In the next stage, the tissue is treated with a sterile solution of0.2-3% (v/v) anionic, non-ionic, zwitterionic or cationic detergent(Triton X-100, Triton X-200, Tween 20, Tween 80, sodium deoxycholate,CHAPS, sodium dodecyl sulfate (SDS), N-lauroyl-sarcosinate, IgepalCA630, Sulfobetain-10 and -16) or chaotropic agent (Tri-n-butylphosphate (TnBP)) prepared in either (i) physiological buffer solution(Hanks' Balanced Salt Solution (HBSS), HEPES, Phosphate Buffered Saline(PBS), Tris-Buffered Saline (TBS)) adjusted to pH=7-9 withanti-microbial agents (penicillin, vancomycin, streptomycin, gentamycin,kanamycin, neomycin, sodium azide (NaN₃)) with or without anti-fungalagents (Amphotericin B, Nystain) or with (ii) 50-70% ethanol, for 24-48hours at T=4-40° C. Afterward, the tissue is rinsed with a sterile 50 mMTris buffer solution adjusted to pH 9 (containing no detergent orchaotropic agent) for 12-24 hours at T=4-40° C.

Next the tissue is treated with a 0.05-3% (v/v) Peracetic Acid (PAA)solution in ethanol or Phosphate Buffered Saline (PBS) neutralized topH=7 to 7.5, for a duration of 30 minutes to 4 hours at T=20-40° C.After, the tissue is rinsed with sterile physiological buffer (Hanks'Balanced Salt Solution (HBSS), HEPES, Phosphate Buffered Saline (PBS),Tris-Buffered Saline (TBS)) with anti-microbial agents (penicillin,vancomycin, streptomycin, gentamycin, kanamycin, neomycin, sodium azide(NaN₃)) with or without anti-fungal agents (Amphotericin B, Nystain) for12-24 hours at T=4-40° C.

Finally, tissues are bottled under sterile conditions in either (i)sterile physiological buffer (Hanks' Balanced Salt Solution (HBSS),HEPES, Phosphate Buffered Saline (PBS), Tris-Buffered Saline (TBS)) withanti-microbial agents (penicillin, vancomycin, streptomycin, gentamycin,kanamycin, neomycin, sodium azide (NaN₃)) with or without anti-fungalagents (Amphotericin B, Nystain) or (ii) 50-70% ethanol and stored atT=4-25° C.

These processes produce decellularized and sterilized tissue. Analysessimilar to those employed in the Examples above and/or known in the artreveal that cell nuclei, major immunogenic cell proteins (e.g., HLA-DRand HLA-A,B,C), and DNA content are substantially reduced compared tocontrols. Further, significant reductions in the cytoskeletal proteinssuch as, e.g., vimentin and Beta-actin, are achieved compared tocontrols. Thermostability analysis of collagen reveals that thedecellularization methods largely preserve the native structure ofcollagen within the tissue. Decellularization by, e.g., Aqueous orEthanol Decellularization protocols, also reduces the amount of bacteria(Staphylococcus aureus, Streptococcus pyogenes (group A strep.),Enterococcus sp., and Bacillus subtilis). Removal of all four bacterialspecies is obtained via inclusion of a 1% (v/v) peracetic acid rinse.The resulting graft is acceptable for transplant according to theguidelines provided by the American Association of Tissue Banks (AATB)standards [AATB (2006); (2008)].

All publications and patent applications cited in this specification areherein incorporated by reference in their entirety for all purposes asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference for allpurposes.

Although the foregoing present disclosure has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this present disclosure thatcertain changes and modifications can be made thereto without departingfrom the spirit or scope of the appended claims.

Tables

TABLE 1 Results of the DNA Assay indicating removal of cellular DNA fromhuman skin samples to below detection levels (0.5 ng/mg dry wt oftissue) after decellularization treatment. DNA Content in Sample ng/mg(dry wt) of tissue Fresh Human Skin 650 ± 131 Aqueous DecellularizationMethod None Detected** Ethanol Decellularization Method None Detected****None detected is <0.25 g/ml or <0.5 ng/mg (ppm) dry wt, the loweststandard detected by the PicoGreen ® DNA Assay (Invitrogen ™ MolecularProbes ®) using a 10 mg dry wt of sample.

TABLE 2 Hydrothermal Isometric Tension (HIT) Test Results HIT Inflection(° C.) or Denaturation Temperature Donor Fresh Aqueous 70% EthanolTD09-163 67.5 68 66 TD09-195 66.5 66.5 66 TD09-196 67 67.5 64.5 TD09-24267 66.5 66.5 Average ± SD 67.0 ± 0.4 67.1 ± 0.8 65.8 ± 0.9 The HIT testis a measure of the stability of collagen within soft tissues. If ourprocess had altered the structure of collagen (the main component inhuman dermis), then we would see a significant drop in the DenaturartionTemperature of the decellularized human skin. The data shown above from4 different donors indicates that there is no change in collagenstability and therefore collagen structure, after decellularizationtreatment.

Di- rect Concentration of Wash solution Plat- Microorganism 10⁻³ 10⁻⁴10⁻⁵ 10⁻⁶ 10⁻⁷ 10⁻⁸ Skin ing Table 3(a) Donor TD10-238 S. Aureus 0 0 0 00 0 0 0 Streptococcus 0 0 0 0 0 0 0 0 Enterococcus 0 0 0 0 0 0 0 0Bacillus 0 0 0 0 0 0 0 0 Negative 0 0 0 0 0 0 0 0 Control Table 3(b)Donor TD10-272 S. Aureus 0 0 0 0 0 0 0 0 Streptococcus 0 0 0 0 0 0 0 0Enterococcus 0 0 0 0 0 0 0 0 Bacillus 0 0 0 0 0 0 0 0 Negative 0 0 0 0 00 0 0 Control Table 3(c) Donor TD09-195 S. Aureus 0 0 0 0 0 0 0 0Streptococcus 0 0 0 0 0 0 0 0 Enterococcus 0 0 0 0 0 0 0 0 Bacillus 0 00 0 0 0 0 0 Negative 0 0 0 0 0 0 0 0 Control Table 3(d) Donor TD10-240S. Aureus 0 0 0 0 0 0 0 0 Streptococcus 0 0 0 0 0 0 0 0 Enterococcus 0 00 0 0 0 0 0 Bacillus 0 0 0 0 0 0 0 0 Negative 0 0 0 0 0 0 0 0 Control

Table 3a-d. Results of bioburden microbiology assay after 7 days ofincubation. Samples of fresh human skin from 4 donors—(a) TD10-238, (b)TD10-272, (c) TD09-195, (d) TD10-240—were each inoculated with 400 μL ofsolution containing 10⁸ CFU/mL of S. aureus, Streptococcus,Enterococcus, or Bacillus. The samples of fresh skin were thendecellularized using the process outlined in FIG. 1. Afterdecellularization, samples were washed with 10 mL of sterile phosphatebuffered saline (PBS) and serial dilutions (10⁻³ to 10⁻⁸) of the PBSwash were spot plated onto Tryptic Soy Agar (TSA plates). The PBS washedskin (labelled “Skin”) and a sample of decellularized skin not washedwith PBS (labelled “Direct Plating”) were also direct plated onto TSAplates. All TSA plates were incubated for 7 days. No colonies ofbacteria were detected in PBS solutions used to rinse the decellularizedskin or by direct plating of decellularized skin. Negative controlsconsisted of TSA plates alone.

Mi- Concentration of Wash solution croorganism 10⁻³ 10⁻⁴ 10⁻⁵ 10⁻⁶ 10⁻⁷10⁻⁸ Skin Table 4(a) Donor TD10-238 S. Aureus TNTC TNTC TNTC 52 9 0 TNTCStrep- TNTC TNTC TNTC 127 12 1 TNTC tococcus En- TNTC TNTC TNTC 72 7 1TNTC terococcus Bacillus TNTC TNTC TNTC 24 2 1 TNTC Table 4(b) DonorTD10-272 S. Aureus TNTC TNTC TNTC 80 11 0 TNTC Strep- TNTC TNTC TNTC 10312 1 TNTC tococcus En- TNTC TNTC TNTC 61 7 1 TNTC terococcus BacillusTNTC TNTC TNTC 33 2 0 TNTC Table 4(c) Donor TD09-195 S. Aureus TNTC TNTCTNTC 59 8 1 TNTC Strep- TNTC TNTC TNTC 90 6 0 TNTC tococcus En- TNTCTNTC TNTC 71 8 1 TNTC terococcus Bacillus TNTC TNTC TNTC 23 1 0 TNTCTable 4(d) Donor TD10-240 S. Aureus TNTC TNTC TNTC 57 6 0 TNTC Strep-TNTC TNTC TNTC 88 4 0 TNTC tococcus En- TNTC TNTC TNTC 88 4 0 TNTCterococcus Bacillus TNTC TNTC TNTC 30 3 1 TNTC

Table 4a-d. Results of microbiology assay for inoculation positivecontrols. Samples of decellularized human skin from 4 donors—(a)TD10-238, (b) TD10-272, (c) TD09-195, (d) TD10-240—were each inoculatedwith 400 μL of solution containing 10⁸ CFU/mL of S. aureus,Streptococcus, Enterococcus, or Bacillus. The samples were washed with10 mL of sterile phosphate buffered saline (PBS) and serial dilutions(10⁻³ to 10⁻⁸) of the PBS wash were spot plated onto Tryptic Soy Agar(TSA plates). The PBS watched skin (labelled “Skin”) was also directplated onto TSA plates. All TSA plates were incubated for 24 hours andthe number of colonies determined. All samples showed positive bacterialgrowth indicating that inoculated bacteria will grow on decellularizedtissue. Note: TNTC=Too Numerous To Count.

TABLE 5 Microorganism Donor S. Aureus Streptococcus EnterococcusBacillus Fungus TD10-238 No Growth No Growth No Growth No Growth NoFungi TD10-195 No Growth No Growth No Growth No Growth No Fungi TD10-272No Growth No Growth No Growth No Growth No Fungi

Table 5. Results of third-party testing for bioburden removal. Samplesof fresh skin (2 cm×2.5 cm) from 3 different donors were each inoculatedwith 400 μL of solution containing 10⁸ CFU/mL of S. aureus,Streptococcus, Enterococcus, or Bacillus. The samples of fresh skin werethen decellularized using the process outlined in FIG. 1. Afterdecellularization, samples were sent to the Department of Pathology andLaboratory Medicine, Capital District Health Authority, Halifax, NovaScotia, Canada for analysis according to the American Association ofTissue Banks (AATB) Standards (12^(th) edition). In all samples, nobacteria or fungi were detected.

REFERENCES

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What is claimed is:
 1. A bioprosthetic tissue, wherein the tissue is95±5% free of nucleic acids, wherein the tissue is 95±5% free of majorhistocompatibility molecules, wherein the tissue is 95±5% free ofStaphylococcus bacteria, Streptococcus bacteria, Enterococcus bacteria,and Bacillus bacteria, or complies with American Association of TissueBanks Standards for Tissue Banking under K2.200, wherein the collagenstructure of the tissue is not altered compared to a fresh controltissue as measured by a Hydrothermal Isometric Tension test, and whereinthe bioprosthetic tissue has been prepared from a human tissue having anepidermal cellular layer with an underlying basement membrane matrixsuch that the human tissue's epidermal cellular layer has been removed,leaving the underlying basement membrane matrix intact in thebioprosthetic tissue.
 2. The bioprosthetic tissue of claim 1, whereinthe tissue is 95±5% free of Staphylococcus aureus bacteria,Streptococcus pyogenes bacteria, Enterococcus bacteria, Bacillussubtilis, and fungus, or complies with American Association of TissueBanks Standards for Tissue Banking under K2.200.
 3. The bioprosthetictissue of claim 1, wherein the tissue is human skin.
 4. Thebioprosthetic tissue of claim 1, wherein the nucleic acid is DNA or RNA,and wherein the tissue has less than 0.5 ng/mg dry weight of tissue ofDNA.
 5. The bioprosthetic tissue of claim 1, wherein the majorhistocompatibility complex (MHC) is HLA-DR, and wherein the tissue is95±5% free of HLA-DR as measured by immunohistochemistry, or wherein theMHC is HLA-A, B, C, and wherein the tissue is 95±5% free of HLA-A, B, Cas measured by immunohistochemistry.
 6. The bioprosthetic tissue ofclaim 1, wherein the tissue is 95±5% free of Vimentin as measured byimmunohistochemistry, and wherein the tissue is 95±5% free of Beta-Actinas measured by immunohistochemistry.
 7. The bioprosthetic tissue ofclaim 1, wherein the tissue comprises elastin as measured by histologyusing Van Gieson stain, and wherein the tissue comprises one or moreproteoglycans as measured by histology using Masson's Trichrome stain.8. The bioprosthetic tissue of claim 1, wherein the collagen structureof the tissue is not altered compared to a fresh control tissue asassessed by thermal stability of collagen.
 9. The bioprosthetic tissueof claim 1, wherein the denaturation temperature of the tissue is notaltered compared to a fresh control tissue, and wherein a denaturationtemperature of the tissue is about 64, 65, 66, 67, 68, or 69° C. asmeasured by a Hydrothermal Isometric Tension test.